Broadly impaired NK cell function in non-obese diabetic mice is partially restored by NK cell activation in vivo and by IL-12/IL-18 in vitro

Sofia E. Johansson1, Håkan Hall1, Jens Björklund1 and Petter Höglund1

1 Microbiology and Tumor Biology Center, Karolinska Institutet, 17 177 Stockholm, Sweden

Correspondence to: P. Höglund; E-mail: petter.hoglund{at}mtc.ki.se
Transmitting editor: G. Trinchieri


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NK cells represent a link between innate and adaptive immunity, and may play a role in regulating autoimmune disorders. We have characterized the NK cell population in non-obese diabetic (NOD) mice. The percentage and absolute numbers of NK cells were similar in NOD and controlMHC-matched B6.g7 mice. However, the capacity of NOD NK cells to mediate natural cytotoxicity as well as FcR- and Ly49D-mediated killing was compromised in vitro, suggesting a defect affecting multiple activation pathways. The defect was neither linked to the NK gene complex nor to the MHC, as determined by comparison with mice congenic for these regions. Introducing the ß2-microglobulin mutation on the NOD background further impaired NK cell function, showing that the compromised cytotoxic capacity in these two strains arises from two independent mechanisms. In vivo rejection responses against tumor cells and against MHC classI-deficient spleen cells were decreased in naive NOD recipients, but restored in mice pre-activated with tilorone, a potent activator of NK cells. In addition, killing of some tumor targets was restored in vitro after activation of NK cells with IL-12 plus IL-18 or with IFN-{alpha}/ß, but not with IL-2. Interestingly, natural killing of RMA-S targets by NOD NK cells could not be restored in vitro, indicating that restoration of killing capacity was only partial. Our data suggest a severe, but partially restorable, killing defect in NOD NK cells, affecting activation through several pathways.

Keywords: activation, cytokine, cytotoxicity, diabetes, NK cell


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NK cells are cytotoxic cells that display spontaneous killing of tumor cells and virally infected cells. In vivo, NK cells mediate protection against viruses and neoplastic cells, and also play a role in rejection of allogeneic grafts (1). Apart from their cytotoxic capacity, NK cells may provide a link between innate and adaptive immunity as they produce multiple immunomodulatory cytokines, e.g. IFN-{gamma}, tumor necrosis factor-{alpha}, transforming growth factor-ß and IL-10 (2,3). Because of this property, NK cells have been suggested to play an immuno regulatory role in organ-specific autoimmune disorders, such as experimental autoimmune encephalomyelitis (EAE) and type 1 diabetes (4,5).

A hallmark of NK cell recognition is the interplay between activating and inhibitory receptors expressed by each NK cell (1,6). The missing self model proposes that in normal situations, activation of NK cells is prevented by the action of inhibitory receptors recognizing MHC class I ligands on surrounding cells (7,8). In situations of viral infection, tumorigenesis and transplantation, however, target cells often fail to display a proper set of self MHC class I, explaining why such cells are often recognized by NK cells (7,8). Inhibitory receptors for MHC class I on NK cells have been identified and extensively studied in several species during the last decade (1,6).

Recently, much interest has been devoted to the signals that control NK cell activation. In contrast to T and B cells, NK cells do not express rearranged receptors ensuring clonal specificity. Instead, activation of individual NK cells relies on the expression of multiple germline-encoded receptors, recognizing a broad spectrum of ligands and requiring different sets of adaptor molecules to convey signals downstream (912). A well-studied NK cell receptor is CD16 (13), that binds the Fc part of Ig and induces antibody-dependent cellular cytotoxicity (ADCC). NK cells also express activating forms of Ly49 (Ly49D, -H -P and -W) and CD94/NKG2 (NKG2C and NKG2E) receptors, recognizing endogenous or virally encoded MHC class I-like ligands (1). Common to CD16 and activating forms of Ly49 and CD94/NKG2 receptors is their association with Ig-like tyrosine-based activation motifs (ITAM)-containing adaptor proteins such as Fc{epsilon}RI{gamma} or DAP12 (1417). Phosphorylation of ITAM leads to the recruitment of the protein tyrosine kinases Syk and ZAP-70 that binds to phosphatidylinositol-3-kinase (PI3K) (12,13,18). Another main activating pathway in NK cells relies on the NKG2D receptor, which recognizes a number of stress-induced and virally expressed ligands (11,19). To signal, NKG2D on NK cells associates mainly with the adaptor protein DAP10 (11). DAP10 does not contain an ITAM, but has the capacity to bind directly to PI3K, thereby bypassing Syk and ZAP-70. After the conjunction of the different pathways at PI3K, the signaling follows a common path towards cytotoxicity, including the PI3K–Rac–PAK–MEP–ERK pathway (20). Apart from these receptors, NK cells also express other receptors with less well-studied function, such as 2B4 and ICOS, which seem to function mainly as co-stimulatory molecules (21,22).

In T and B cell activation, ITAM-dependent signals are critical (9). In contrast Syk and ZAP-70, and hence supposedly ITAM, are not necessary for NK cell cytotoxicity against several tumor cells (23). Some of those targets may be recognized by NKG2D, as was recently reported for the prototype NK cell target YAC-1 (24). However, other tumor cells, such as the MHC class I-deficient lymphoma RMA-S, are efficiently killed by NK cells in the absence of ITAM signaling capacity (23) and despite their lack of NKG2D ligands (19). Hence, yet other modes of activation must exist in NK cells.

NK cells have been implicated to play both pathological and protective roles in autoimmune diseases such as EAE, experimental autoimmune myasthenia gravis (EAMG), Inflammatory bowel disease and virally induced type 1 diabetes (4,5,2527). The non-obese diabetic (NOD) mouse is a model of type 1 diabetes (28). Several studies have reported decreased NK activity against the target cell YAC-1 in NOD mice (2931). NOD-SCID mice have also been used for human xenograft transplantation, much due to their low NK activity (32). In this paper, we report an extensive functional characterization of NOD NK cells in vitro and in vivo. Our data points to a broad intrinsic functional deficiency in NOD NK cells that extends beyond deficient NKG2D-dependent YAC-1 killing (33) and affects several activation pathways. In addition, the killing capacity could be restored, at least against some targets, after NK cell activation in vivo, and after stimulation with IL-12 and IL-18 in vitro, suggesting a dynamic control of NK cell activity in this mouse strain.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell cultures
YAC-1, RMA, RMA-S, IC-21, Ba/F3 and CHO cells were grown in RPMI 1640 medium supplemented with 5% FCS, 2 mM L-glutamine, 10 IU/ml penicillin and 10 µg/ml streptomycin (Life Technologies, Paisley, UK). In the culture of the murine hematopoietic cell line Ba/F3, 5–10 ng/ml IL-3 was added. The adherent murine macrophage-like cells IC-21 and CHO cells were detached by incubation for 5–7 min in PBS with 0.5 mM EDTA, and washed before use. YAC-1, RMA and RMA-S are T cell lymphomas of B6 origin. For cultures with IL-2 or IL-12 plus IL-18, spleens were dispersed and the erythrocytes were lysed. Then, 2.5–3 x 106 cells/ml were cultured for 4 days in {alpha}-MEM containing 10% FCS, HEPES, penicillin–streptomycin, 0.1% ß-mercaptoethanol with the addition of either 1000 U/ml human rIL-2 [lymphokine-activated killer (LAK) cultures] or 1 ng/ml IL-12 and 100 ng/ml IL-18 (PeproTech, London, UK). For IFN stimulation, spleens were dispersed and erythrocytes lysed. Then, 2 x 106 cells/ml were incubated for 4 h in 37°C in RPMI medium with additives as above plus 200 U/ml IFN-{alpha} and 200 U/ml IFN-ß (R & D Systems, Abingdon, UK).

Mice
NOD/Lt (referred to as NOD throughout this study), NODß2m, NODNK1.1 [congenic for the B6 NK complex (NKC) (31)], C57BL/6 (B6), B6ß2m and B6.g7 mice were kept and bred at the animal facility at the Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm. All mice were 5–9 weeks old, and were sex- and age-matched (within 2 weeks) in each experiment. When indicated, mice were given 2 mg tilorone analogue R10,874DA orally 1 day before the experiment (T-8014; Sigma-Aldrich, Stockholm, Sweden).

Antibodies and FACS analysis
For NK cell depletion, 100 µg PK136 (anti-NK1.1, mouse IgG2a) or 200 µg TMß-1 (anti-IL-2Rß, rat IgG2b) were given i.p. 2 days before the experiment. PK136 and TMß-1 were purified from hybridoma supernatants using a Protein G column. For FACS analysis and sorting, the TMß-1 antibody was conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR). Dx5–phycoerythrin (PE), CD3–PerCP, CD3–PE (145-2C11), CD11b–FITC, CD43–PE and IFN-{gamma}–PE were purchased from PharMingen (Stockholm, Sweden). NK1.1–allophycocyanin was a kind gift from James P. Disanto (Paris, France). To enumerate NK cells, spleens were erythrocyte lysed and incubated for 1 h in cell culture medium containing 1.5 U/ml collagenase P (Roche Diagnostics, Mannheim Germany).

Cytotoxicity assays
Cytotoxicity was measured using a standard 51Cr-release assay. As effector cells, we used freshly isolated splenocytes, or sorted TMß-1+CD3 cells, from tilorone-treated mice. Alternatively, cytokine-stimulated cultures (see cell cultures) were used. As targets, 51Cr-labeled tumors as mentioned in figures were used. For ADCC, 51Cr-labeled RMA cells were incubated for 10 min with an antibody against the T cell-surface antigen Thy-1.2 (30-H12) at a final concentration of 0.1 or 0.3 µg/ml and washed before adding effectors. E:T ratios are indicated in the figures.

Conjugate assay
LAK cultures from NOD and B6.g7 mice were labeled with 0.1 µM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) as described below. YAC-1, RMA and RMA-S were incubated for 15 min with the dialkylcarbocyanide DiD (Molecular Probes) at 37°C and washed in RPMI. All the cells were incubated in 37°C for 20 min. Approximately 200,000 effector and target cells were mixed, centrifuged at 300 r.p.m. (~18 g) for 3 min and then incubated at 37°C for 15 min. Paraformaldehyde was added to a final concentration of 2% and the cells were incubated for 15 min at room temperature. After fixation, the conjugates were labeled with TMß-1 to exclude non-NK cells from the analysis and the samples were run in the FACS.

CFSE staining and in vivo killing of CFSE-labeled cells
Spleen cells were erythrocyte lysed, filtered through a 70-µm cell dispenser, washed and resuspended in PBS supplemented with 0.3 or 3 µM of the dye CFSE. The cells were incubated at 37°C for 10 min and washed in RPMI medium containing 5–10% FCS. Then, 1 x 107 of each cell type was mixed and the mixture subsequently injected i.v. suspended in 200 µl PBS. After 18–24 h, the spleens were taken out, erythrocytes lysed and the relative percentage of cells in each CFSE population was measured in a FACScan (BD Biosciences, Mountain View, CA). Ascites-grown RMA and RMA-S cells were stained using 0.6 or 6 µM of CFSE. The higher concentration of CFSE was not toxic to the cells, as the survival was not decreased after incubation (data not shown) and the results were reproducible when either of the cell populations were stained with the higher concentration. 1.5 x 107 tumor cells of each type were mixed and injected as above. After 16–20 h the lungs were removed and incubated in 37°C for 1 h in RPMI with 400 U/ml collagenase type IV (Sigma-Aldrich). Erythrocytes were lysed and the resulting cell population was filtered through a 70-µm cell dispenser followed by FACScan analysis. The relative survival of CFSEhigh cells compared to CFSElow cells was calculated as follows: (acquired number of CFSEhigh cells in sample/acquired number of CFSElow cells in sample)/(acquired number of CFSEhigh cells in injection mix/acquired number of CFSElow cells in injection mix). At least 3000 CFSE+ cells were acquired in each sample.

Intracellular IFN-{gamma} staining
Spleens from two tilorone-treated mice in each group were erythrocyte lysed and pooled. Dx5+ cells were sorted by MidiMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Between 50 and 60% of the purified cells were NK cells. Samples of 1–2 x 105 bead-coated cells were incubated for 5 h in a 96-well plate in RPMI medium containing 0.67 µl/ml GolgiStop alone or in the presence of either YAC-1 cells (in a 1:1 ratio) or 50 ng/ml of phorbol myristate acetate (PMA) and 100 ng/ml ionomycin. Alternatively, splenocytes were cultured with IL-12 and IL-18 as described above. On day 4, GolgiStop was added during the last 5 h of culture. Intracellular staining for IFN-{gamma} was performed according to protocol with Cytofix/Cytoperm Plus kit (PharMingen). A combination of TMß-1 and CD3 antibodies was used as surface markers to detect NK cells.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The NK cell populations in NOD and B6.g7 mice are of similar sizes
Previous enumerations of NK cells in NOD mice were performed in NOD mice congenic for the NK gene complex from B6 (27,28). In order to exclude an effect of the B6 NKC on the size of the NK population, we evaluated the use of Dx5 [recognizing the CD49b integrin (34)] together with TMß-1 [binding to the ß chain of the IL-2 receptor (32)] as markers for NOD NK cells. Control stainings, performed in B6 mice, revealed that ~90% of the CD3NK1.1+ population were TMß-1+DX5+ (Fig. 1A and B) and that ~97% of CD3TMß-1+Dx5+ cells expressed NK1.1 (Fig. 1C and D). From this result, we conclude that the CD3TMß-1+Dx5+ cell population adequately reflects the number of NK cells in NOD mice.



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Fig. 1. The size of the NK cell population is similar in NOD and B6.g7 mice. All plots are gated on lymphocytes. (A–D) Spleen cells from a B6 mouse were stained with TMß-1–FITC, Dx5–PE, CD3–PerCP and NK1.1–allophycocyanin. (A) The dot-plot shows CD3 versus NK1.1 staining. (B) Of NK1.1+CD3 cells in the R2 gate in (A), 90.5% fall into the Dx5+ TMß-1+ population. (C) The plot is gated on CD3 lymphocytes, showing Dx5 versus TMß-1. Note the Dx5+TMß-1 population. (D) Histogram showing NK1.1+ cells in the R3 gate in (C), 97.3% of these cells are NK1.1+. (E) Average percentage of CD3TMß-1+Dx5+ cells in the lymphocyte gate from five mice in each group. Mean percentage of NK cells in B6.g7 was 2.14% and in NOD 2.73%. Bars represent a 95% confidence interval.

 
We applied this staining on NOD spleen cells and compared the result with control B6.g7 mice, which are B6 mice congenic for the NOD MHC, the main diabetes susceptibility locus of NOD mice (36,37). The fraction of NK cells in the spleen of NOD mice was 2.73% and in B6.g7 2.14% (Fig. 1E). In absolute numbers, we found 1.7 x 106 (±0.73 x 106) NK cells in NOD spleen (n = 4) and 1.4 x 106 (±0.04 x 106) in B6.g7 (n = 2). Thus, the pools of NK cells are of similar sizes in NOD and MHC-matched B6 mice.

NOD NK cells are defective in killing of several tumors and produce less IFN-{gamma} after YAC-1 co-culture
We extended earlier work, indicating a NOD NK cell defect in cytotoxic potential in vitro (2831), by analyzing a larger panel of tumor targets. As effector cells we used in vivo activated splenocytes from mice treated with a tilorone analogue, a potent NK activator (38). B6 and B6.g7 mice were used as controls. Our result shows that NOD splenocytes were deficient in killing of YAC-1 cells compared to B6 splenocytes (Fig. 2A). In addition, killing was decreased against RMA, RMA-S, Ba/F3 and IC-21 (Fig. 2B–E). B6 and B6.g7 mice displayed similarly high cytotoxicity, showing that the NK cell activity was not affected by the NOD MHC by itself (Fig. 2A–E).



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Fig. 2. The NOD defect is evident against multiple tumor targets and is inherent to the NK cell. (A–E) Cytotoxicity against five different tumor cell targets obtained in a 4-h 51Cr-release assay using freshly isolated spleen cells from tilorone-activated mice as effector cells. Data is an average of two mice in one representative experiment out of three to seven. The x-axis labels indicate the E:T ratios. (F) The NOD deficiency is intrinsic to NK cells. Spleen cells from tilorone-activated mice were nylon wool passed and TMß-1+CD3 cells were sorted by FACS. Purity was 87–97%. One of two identical experiments. (G) Mac-1 expression on NK cells. The plot is gated on CD3 lymphocytes. In this experiment, out of TMß-1+CD3 cells, 66% were Mac-1+ in NOD and 77% in B6.g7. (H) CD43 expression on NK cells. The plot is gated on CD3 lymphocytes. All TMß-1+ cells were CD43+. (I) IFN-{gamma} release in NOD NK cells was lower after stimulation with YAC-1. Mice were treated with tilorone in vivo and Dx5+ cells from spleens were sorted with magnetic beads. Cells were co-cultured with YAC-1 in a 1:1 ratio and IFN-{gamma} production was measured by intracellular staining. Plots are gated on TMß-1+CD3 cells. One representative experiment of three.

 
The decreased cytotoxicity could be due to inhibitory effects from other cells upon the NK cell population. To investigate this possibility, we sorted NK cells from tilorone-treated NOD and B6.g7 mice, and used them directly as effectors. Sorting did not correct the reduction in NOD cytotoxic activity against YAC-1 cells (Fig. 2F), showing that the functional deficiency, at least against this target, was intrinsic to the NK cell population. To test the possibility that the decrease in cytotoxicity was a consequence of an impaired capacity to bind to their target cells, we performed conjugation assays with NOD and B6.g7 IL-2-stimulated NK cells against YAC-1, RMA and RMA-S targets. The results showed that NOD and B6.g7 NK cells were equally efficient at forming conjugates with all these targets (data not shown), excluding this possibility.

Previous work has suggested that expression of certain integrins, including Mac-1 (CD11b or {alpha}Mß2) and CD43 (leucosialin), identifies mature NK cells with fully developed capacity to secrete IFN-{gamma} and to mediate cytotoxicity (39). To test whether the functional deficiency of NOD NK cells was a consequence of deficient NK cell maturation, we compared the expression of Mac-1 and CD43 on CD3TMß-1+ NOD and B6.g7 spleen cells. In both strains, we found that Mac-1 was expressed on most CD3TMß-1+ cells and that CD43 was present on virtually all of those cells (Fig. 2G and H). We did observe a small, but statistically significant (P <= 0.01), reduction in the percentage of CD3TMß-1+Mac-1+ cells in NOD compared to B6.g7 mice when the mean of all experiments was calculated (61 ± 5.7 versus 73 ± 5.4). However, we find it unlikely that this small reduction in Mac-1+ cells could explain the full magnitude of functional deficiency in NOD NK cells, especially since all of the cells were simultaneously positive for CD43. We thus conclude that the NK cell defect in NOD mice is unlikely to be explained by a defect in NK cell maturation.

We next tested cytokine secretion after co-culture of in vivo stimulated NK cells with YAC-1. Dx5+ spleen cells from tilorone-treated mice were purified with magnetic beads and mixed in a 1:1 ratio with YAC-1 cells. After 5 h of co-culture, 30.8% of B6 NK cells (TMß-1+CD3) produced IFN-{gamma}, while only 20.7% of NOD NK cells did so (Fig. 2I). This reduction by one-third correlated well with the diminished NOD cytotoxicity against YAC-1 (Fig. 2A and F) and with recently published data with IL-2-activated NOD NK cells (33). Thus. cytotoxicity and cytokine release are both compromised in NOD NK cells against this target. However, we found no differences in the ability of NOD NK cells to produce IFN-{gamma} after stimulation with PMA and ionomycin (data not shown), demonstrating that the IFN-{gamma}-secreting machinery in NOD NK cells is functional if properly stimulated.

The NOD defect affects activation through several activating pathways
We next tested the killing capacity against targets for which the activating receptors and signaling pathways are better characterized and distinct from the ones used for recognizing the tumors in Fig. 2. To trigger ADCC via CD16, RMA target cells were incubated with an antibody against the Thy-1.2 antigen followed by exposure to IL-2-activated NK cells. Our experiment showed that B6.g7 effectors strongly increased their killing after addition of anti-Thy-1.2 antibody, while NOD NK cells showed a more moderate increase (Fig. 3A). Similarly, CHO cells, which are killed by NK cells mainly via activation of the Ly49D receptor signaling through the adaptor molecule DAP12 (4042), were killed much more efficiently by B6.g7 NK cells compared to NOD NK cells, suggesting that triggering via Ly49D/DAP12 may also be impaired in NOD NK cells (Fig. 2B). Thus, Figs 2 and 3 together show that NOD NK cell killing was generally impaired, being manifested in cytotoxicity against YAC-1 [NKG2D(22)], RMA and RMA-S (unknown activating receptor), as well as in CD16- and Ly49D-mediated activation.



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Fig. 3. The NOD defect is evident in ADCC and Ly49D-dependent killing. (A) ADCC. LAK cells in a 51Cr-release assay against RMA cells coated with two different doses of anti-Thy-1.1 antibody. The E:T ratio was 30:1. One representative experiment of five. (B) Killing of CHO cells by NOD and B6.g7 LAK cells. One experiment of two.

 
The NK deficiency is not linked to the NK gene complex and is distinct from the NK cell defect in ß2m mice
We decided to evaluate the cytotoxic capacity of the NOD mouse congenic for the B6 NKC in our mouse colony, and compare it to NOD and B6. In agreement with recently published data (33), the cytotoxic capabilities against YAC-1 targets by IL-2-activated NK cells from NOD and NOD.NK1.1 mice were indistinguishable. In addition, no differences were found between NOD and NOD NK1.1 mice against RMA-S (Fig. 4A) or CHO (data not shown) with the same effector cells. We therefore confirm and extend previous data by showing that the NK cell defect in NOD mice is not linked to the NKC in a way that was detectable using IL-2-activated effector cells.



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Fig. 4. The NOD NK defect is not linked to the NK1.1 complex and is complementary to the decreased activity seen in ß2m mice. Cytotoxic assay with LAK cultures of NOD, NOD.NK1.1 and B6. Cytotoxicity against (A) YAC-1 and (B) RMA-S. (C) The cytotoxic defects in NOD and ß2m mice are additive. YAC-1 killing by freshly isolated splenocytes from tilorone-activated mice of the indicated strains. One of three to five experiments.

 
NK cells from ß2m mice are functionally deficient in killing capacity against several tumor cells (43,44). Given the similarities in reduced tumor cell killing, we asked whether NOD and ß2m mice had the same basis for their reduced NK cell capacity. Our result showed that the reduction in killing capacity in NOD and ß2m mice was additive. While B6ß2m and NOD NK cells showed intermediate killing, NODß2m NK cells were almost completely unable to kill YAC-1 targets (Fig. 4B). Since the defects were cumulative, the molecular bases for NK cell deficiency in NOD and ß2m mice must be different and complementary. The fact that NOD recipients reject NODß2m grafts (Fig. 6A) and that NODß2m recipients are themselves unable to reject such grafts (data not shown) further support this notion.



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Fig. 6. The NOD defect is NK dependent and restored in vivo with tilorone treatment. All recipients were pre-activated with the tilorone analogue T-8014. NK cells were depleted in vivo with TMß-1 antibody where indicated. (A) Rejection of ß2m spleen cells (see Fig. 5 for method) in NOD and B6 mice. (B) Rejection of RMA-S in NOD and B6.g7 mice. The experiment was repeated several times, with both B6 and B6.g7 mice as controls. Error bars show SD.

 
Rejection of ß2m splenocytes and tumors in vivo is reduced in NOD mice, but is increased to control levels after treatment of the recipients with tilorone
We used a novel method to test the in vivo rejection response in NOD mice. The set-up is depicted in Fig. 5(A). In essence, the method allows determination of the relative elimination of an NK-sensitive target compared to an NK-resistant target. The two target cell populations are labeled with different concentrations of CFSE (45,46) and inoculated into the same recipients. By determining the ratio between the remaining cell number in the two populations in the spleen, a measure of the in vivo NK activity is obtained.



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Fig. 5. Rejection in vivo is reduced in NOD mice. (A) The method for studying NK cytotoxicity in vivo. MHC+ and MHC cells were labeled with different concentrations of CFSE, mixed, and injected i.v. into NOD and B6.g7 recipients. The spleens or lungs were taken out 18 h later. The ratio between CFSEhigh and CFSElow cells was determined by FACScan. The relative rejection of MHC cells was determined by comparing this ratio to that of the inoculation mixture, determined at the start of the experiment. (B) CFSE-labeled B6 and B6ß2m spleen cells (107) were injected i.v. into naive B6 mice, and NOD and NODß2m cells into NOD mice. The bars show the relative number of ß2m cells retrieved in the spleen after 18 h, compared to injected B6 or NOD cells respectively. Bars show an average of four mice in each group. Error bars show the 95% confidence interval. B6 were used as control recipients for the ß2m grafts (instead of B6.g7 mice) since the ß2m mouse is made on this background. P < 0.01 using a paired t-test. (C) Rejection of RMA-S in naive NOD and B6.g7 mice. The difference between NOD and B6 at 18 h was statistically significant (P < 0.05) using a paired t-test including three experiments. The diagram shows an average of three different experiments with two mice in each group per experiment. Error bars show SD.

 
As expected from a previously published study (30), naive NOD mice were significantly less effective than B6 mice in rejecting ß2m cells (Fig. 5B). We next tested the in vivo rejection of a tumor target cell in NOD recipients: the MHC lymphoma RMA-S. As an internal control in this case we used its MHC+ and NK-resistant counterpart, RMA. Tumor cells preferentially localize to the lung after i.v. inoculation [(47) and data not shown] and we therefore measured the eliminated fraction of tumor cells in this organ. Supporting our in vitro data, we observed that RMA-S cells were less efficiently rejected in untreated NOD mice compared to B6.g7 (Fig. 5C; P < 0.05), even though the low ratio between RMA-S and RMA revealed a surprisingly efficient rejection response in both recipients.

To test if the rejection responses could be affected by NK cell activation in vivo, we pre-treated the recipients with a tilorone analogue, a known augmentor of NK cell activity, before grafting. Interestingly, NOD mice that had received a single dose of tilorone 1 day before grafting rejected both NODß2m cells and RMA-S tumor cells (Fig. 6A and B respectively). Elimination of TMß-1+ cells completely prevented rejection of both NODß2m spleen cells and RMA-S lymphoma cells, demonstrating that NK cells were indeed responsible for the rejection (Fig. 6A and B).

The deficiency of NOD NK cells is partially restored after stimulation with IL-12/IL-18 and IFN-{alpha}ß in vitro
The surprisingly efficient NK responses in vivo, particularly in tilorone-treated mice, suggested that the NOD NK cell activity could be restored under physiological conditions. To investigate if the defect could be overcome in vitro using other types of stimulations than IL-2 [that did not correct the defect; Fig. 7A and (33)], we tested the killing capacity of NOD NK cells activated directly with either a combination of IL-12 and IL-18 or with IFN-{alpha}ß (48). Our results showed that IL-12 and IL-18 virtually restored NOD NK cell cytotoxicity up to B6.g7 levels against YAC-1 and CHO (Fig. 7A and B). Interestingly, killing of RMA-S was still defective (Fig. 7B). IL-12 and IL-18 together also triggered a similar spontaneous IFN-{gamma} release in NK cells from the two strains (Fig. 7D). Similarly, 4 h of in vitro stimulation with IFN-{alpha}ß led to a substantial increase in NOD NK cell cytotoxicity against YAC-1 compared to NK cells isolated from tilorone-activated mice. However, cytotoxicity was still somewhat lower compared to IFN-stimulated B6.g7 NK cells (Fig. 7C), suggesting only partial restoration. As for IL-12 and IL-18, cytotoxicity against RMA-S cells was lower by NOD than by B6.g7 splenocytes after IFN treatment, while cytotoxicity against CHO was similar, but rather low (~10%), by both NOD and B6.g7 cells (data not shown). In conclusion, stimulation with cytokines other than IL-2 in vitro could partially restore the NOD NK cell cytotoxic activity, in some cases virtually up to control levels.



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Fig. 7. IL-12 plus IL-18 or IFN-{alpha} plus IFN-ß treatment partially restore the NOD NK defect. (A and B) Cytotoxicity of IL-2 or IL-12 + IL-18 cultures. (A) Effector cells were cultured with cytokines for 4 days, and then TMß-1+ CD3 cells were sorted out and used in a cytotoxicity assay against YAC-1. (B) Unsorted cytokine cultures against RMA-S and CHO, adjusted for the number of NK cells in the culture. The x-axis shows the number of NK cells (TMß-1+ CD3) per target cell in the assay. This adjustment was necessary because the percentage of NK cells differed between IL-2 and IL-12 + IL-18 cultures. (C) Cytotoxicity of IFN-{alpha} + IFN-ß-stimulated cultures compared to tilorone-stimulated splenocytes. Splenocytes from naive mice were stimulated for 4 h with IFN-{alpha} + IFN-ß or freshly isolated from mice stimulated with tilorone as in Fig. 1. (D) IFN-{gamma} production in IL-12 + IL-18-stimulated NK cells from NOD is normal. IFN-{gamma} production was measured by intracellular staining. Plots are gated on TMß-1+CD3 cells. One representative of at least three experiments.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We found in this study that NOD NK cells failed to kill all tested targets, irrespective of whether they triggered lysis in an ITAM-dependent or -independent manner. This makes NOD NK cells different from NK cells in knockout mice with deficiencies in single NK cell receptors, adaptors or signaling molecules, where the defects are more restricted. For example, mice lacking functional DAP12 molecules fail to kill CHO cells (15), but kill several other targets with normal efficiency (15,49,50). Moreover, Colucci et al. demonstrated that NK cells lacking the two protein tyrosine kinases Syk and ZAP-70 were defective in ITAM-dependent cytotoxicity, but still killed many targets, such as RMA-S, YAC-1 and Ba/F3, with unimpaired efficiency (23). Mice deficient in the signaling molecules Fyn and Vav show quite distinct NK cell defects. Fyn–/– mice have normal killing of YAC-1, IC-21 and CHO, but low killing of RMA-S and ß2m concanavalin A blasts (51). Vav–/– NK cells in turn show normal levels of YAC-1 killing and ADCC after IL-2-activation, while RMA-S killing is still low (52). Thus, deficiencies of individual proximal signaling molecules are unlikely to explain the entire NOD defect. The broad nature of the NOD defect would rather imply either a combination of defects, potentially including one or several of the ones mentioned above, or a deficiency in a single molecule common to all tested activating pathways. The latter alternative includes defects in common signaling components as well as in broadly acting mechanisms such as lytic granules or adhesion molecules. Our data showing that not only cytotoxicity, but also IFN-{gamma} secretion, was impaired after tumor cell encounter argues against a deficiency of lytic granules in NOD NK cells. In addition, adhesion to tumor cells was similar in NOD and B6.g7 NK cells, making an adhesion defect in NOD NK cells less likely. Yet a third alternative is an increased expression of an unknown inhibitory receptor recognizing non-MHC ligands. Although we cannot formally exclude any one of those alternative explanations, we favor a signaling defect in NOD NK cells, affecting one or several signaling components and resulting in a broad down-regulation of effector functions. In this respect, it was interesting to compare NOD mice with mice lacking ß2m; the latter have a broad NK cell defect similar to NOD mice (40,41). An important conclusion from that comparison was that the defects in NOD and ß2m–/– NK cells were not identical, arguing that there are several ways to broadly down-regulate NK cell effector functions.

It was recently demonstrated that upon NK cell activation, NKG2D expression is strongly up-regulated on B6 NK cells, in turn leading to enhanced NK cell killing against targets expressing NKG2D ligands (53,54). In striking contrast to those data, NOD mice fail to up-regulate NKG2D after activation in IL-2, providing an explanation for the low killing of targets expressing NKG2D ligands (33). Our data are consistent with those conclusions when it comes to the low YAC-1 killing mediated by tilorone-treated and IL-2-activated NOD NK cells. However, most other targets tested in this study, including tumor cells such as CHO and RMA-S as well as ß2m-deficient spleen cells, lack ligands for NKG2D. NKG2D down-regulation per se can therefore not explain the whole defect, but may be only one of several molecular defects in NOD NK cells. However, it cannot be excluded that down-regulation of NKG2D would result in a broad functional NK cell defect. It has been suggested that the balance between activating and inhibitory signals during NK cell development influences the activation threshold in mature NK cells (43,44,51,52,55,56). Perhaps signaling through NKG2D could similarly influence the activation threshold of the responding NK cell, resulting in a state of anergy. Such an anergic state could potentially be induced during NK cell maturation or during activation of mature NK cells that themselves express NKG2D ligands (33). Future studies will be needed to test this possibility.

The partial restoration of NOD NK cell lysis after IL-12 plus IL-18 stimulation was intriguing. We could think of at least two ways in which these cytokines could restore killing by NOD NK cells. First, the cytokines could up-regulate expression of specific molecules that are potentially down-regulated in NOD NK cells and by this make lysis of target cells more efficient. Up-regulation of NKG2D is one possibility to explain increased YAC-1 killing. As already mentioned, IL-2 fails to up-regulate NKG2D on NOD NK cells because the NKG2D ligand is concomitantly expressed on surrounding cells, leading to NKG2D down-modulation (32). One possibility is that IL-12 and IL-18 would fail to induce the NKG2D ligand in the same way as IL-2 (33), allowing NKG2D up-regulation and YAC-1 killing. Alternatively, IL-12 and IL-18 could up-regulate DAP12, an adaptor molecule that is critical for killing of CHO, but which can also associate with the short form of NKG2D and by this stimulate killing of NKG2D ligand-positive targets (53,54). An increase in the expression level of DAP12 would be consistent with the restoration of killing against both CHO and YAC-1. It would also be consistent with the lack of enhancing effects against RMA-S, against which neither NKG2D nor DAP12 are used.

A totally different, but mutually non-exclusive, explanation for how IL-12 and IL-18 could restore the defect is by induction of an alternative activation pathway, or mode of killing, circumventing the defect (and in control NK cells conveying additional activity to existing active pathways). One alternative could be up-regulation of Fas ligand, which has been shown to be induced on NK cell after activation in IL-12 and IL-18 (57,58). This explanation would be consistent with a lack of enhancing effects against RMA-S targets, since this cell type does not express Fas under in vitro conditions (57). In this respect, it is interesting that in vivo passage up-regulates Fas on RMA-S cells in parallel with increased sensitivity to FasL-mediated killing (59). We used in vivo passaged RMA-S cells in our in vivo experiments, which could explain why rejection of RMA-S was efficient in vivo in NOD mice pre-activated with tilorone (60).

Previous studies have shown that IFN{alpha}/ß activates NK cells in vivo, and by this strongly enhances rejection responses against bone marrow grafts and tumor cells (6163). Since rejection responses were increased in tilorone-treated NOD mice, we hypothesized that IFN-{alpha}/ß would restore killing in NOD NK cells in a similar way as IL-12 and IL-18. Indeed, IFN-{alpha}/ß-stimulated NOD spleen cells efficiently killed YAC-1 targets, suggesting that killing could be restored under conditions of IFN-{alpha}/ß stimulation. At the same time, however, these data provide a paradox in that ex vivo NK cells from tilorone-treated NOD mice (in which IFN-{alpha}/ß is suggested to be the main NK cell activator) failed to kill YAC-1 cells in the same 4-h assay. We have no explanation for this intriguing paradox at the moment, but this result suggests that NK cell activation by IFN inducers is a complex event that cannot be directly compared with the direct effects of IFN-{alpha} in vitro. Importantly, this complexity cannot be seen in normal mice, since both tilorone in vivo and IFN-{alpha}in vitro augment tumor cell killing to the same extent. Thus, the NOD defect provides a useful platform for further dissections of the events that are responsible for NK cell ‘priming’ in vivo.

Can NK cells be important in diabetes development? It has been demonstrated that NK cells play a protective role in EAE (5), and a similar role for NK cells in NOD mice would be consistent with the association between diabetes development and an NK cell defect. If the NK cell defect is critical to diabetes development in NOD mice, it should also be possible to prevent diabetes by augmenting NK activity in vivo. Interestingly, poly I:C, a potent IFN inducer and activator of NK cells, protects NOD mice from diabetes (64). Similar results were obtained in NOD mice treated with IL-18 (65), providing an interesting link to our results on restoration of NK activity in the presence of this cytokine. Thus, it appears that an inverse correlation exists between NK activity and diabetes incidence. However, the situation is likely more complex since other reports have suggested a pathogenic rather than protective role for NK cells in autoimmunity, e.g. in EAMG (25). In addition, in a virally induced model of diabetes, NK cells were suggested to promote disease by direct killing of ß cells (4). Thus, the role of NK cells in diabetes is unclear and conclusive results will have to await the generation of NOD mice made selectively deficient for NK cells through gene targeting or antibody depletions.


    Acknowledgements
 
We wish to thank Margareta Hagelin and Maj-Britt Alter for help with in vivo experiments and handling of mice, and Birgitta Wester for help with cell sorting. Thanks also to all members of the Petter Höglund and Klas Kärre groups for advice and helpful discussions. A special thanks to Jakob Michaëlsson and Linda Öberg for help with establishing some of the methods, and to Jacques Zimmer, Werner Held and Claude Carnaud for helpful discussions and critical comments on this manuscript. Claude Carnaud is also gratefully acknowledged for providing the NODNK1.1 mice. This work was supported by grants to P. H. from the Swedish Research Council, Swedish Cancer Society, Cancer Research Institute (USA), Swedish Medical Doctors Association, Torsten and Ragnar Söderberg Foundation, Clas Groschinsky Memory Foundation, Foundation for Scientific work in Diabetology and Karolinska Institute Research Funds. H.H. was supported by a PhD student fellowship from the Swedish Foundation for strategic research.


    Abbreviations
 
ADCC—antibody-dependent cellular cytotoxicity

B6—C57Bl/6

CFSE—carboxyfluorescein diacetate succinimidyl ester

EAE—5,6-experimental autoimmune encephalomyelitis

EAMG—experimental autoimmune myasthenia gravis

ITAM—Ig-like tyrosine-based activation motifs

LAK—lymphokine-activated killer

NKC—NK complex

NOD—non-obese diabetic

PE—phycoerythrin

PI3K—phosphatidylinositol-3-kinase

PMA—phorbol myristate acetate


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Yokoyama, W. M. 1999. Natural killer cells. In Paul, W. E., ed., Fundamental Immunology, 4th edn, p. 575. Lippincott-Raven, Philadelphia, PA.
  2. Kos, F. J. 1998. Regulation of adaptive immunity by natural killer cells. Immunol. Res. 17:303.[ISI][Medline]
  3. Kelly, J. M., Takeda, K., Darcy, P. K., Yagita, H. and Smyth, M. J. 2002. A role for IFN-gamma in primary and secondary immunity generated by NK cell-sensitive tumor-expressing CD80 in vivo. J. Immunol. 168:4472.[Abstract/Free Full Text]
  4. Flodström, M., Maday, A., Balakrishna, D., Cleary, M. M., Yoshimura, A. and Sarvetnick, N. 2002. Target cell defense prevents the development of diabetes after viral infection. Nat. Immunol. 3:373.[CrossRef][ISI][Medline]
  5. Zhang, B., Yamamura, T., Kondo, T., Fujiwara, M. and Tabira, T. 1997. Regulation of experimental autoimmune encephalomyelitis by natural killer (NK) cells. J. Exp. Med. 186:1677.[Abstract/Free Full Text]
  6. McQueen, K. L. and Parham, P. 2002. Variable receptors controlling activation and inhibition of NK cells. Curr. Opin. Immunol. 14:615.[CrossRef][ISI][Medline]
  7. Kärre, K., Ljunggren, H.-G., Piontek, G. and Kiessling, R. 1986. Selective rejection of H-2-deficient lymphoma variant suggest alternative immune defence strategy. Nature 319:675.[ISI][Medline]
  8. Lunggren, H.-G. and Kärre, K. 1990. In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol. Today 11:237.[CrossRef][ISI][Medline]
  9. Colucci, F., Di Santo, J. P. and Leibson, P. J. 2002. Natural killer cell activation in mice and men: different triggers for similar weapons? Nat. Immunol. 3:807.[CrossRef][ISI][Medline]
  10. Jiang, K., Zhong, B., Gilvary, D. L., Corliss, B. C., Hong-Geller, E., Wei, S. and Djeu, J. Y. 2000. Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells. Nat. Immunol. 1:419.[CrossRef][ISI][Medline]
  11. Wu, J., Song, Y., Bakker, A. B., Bauer, S., Spies, T., Lanier, L. L. and Phillips, J. H. 1999. An activating immunoreceptor complex formed by NKG2D and DAP10. Science 285:730.[Abstract/Free Full Text]
  12. Bonnema, J. D., Karnitz, L. M., Schoon, R. A., Abraham, R. T. and Leibson, P. J. 1994. Fc receptor stimulation of phosphatidylinositol 3-kinase in natural killer cells is associated with protein kinase C-independent granule release and cell-mediated cytotoxicity. J. Exp. Med. 180:1427.[Abstract]
  13. Perussia, B. 2000. Signaling for cytotoxicity. Nat. Immunol. 1:372.[CrossRef][ISI][Medline]
  14. Smith, K. M., Wu, J., Bakker, A. B., Phillips, J. H. and Lanier, L. L. 1998. Ly-49D and Ly-49H associate with mouse DAP12 and form activating receptors. J. Immunol. 161:7.[Abstract/Free Full Text]
  15. Tomasello, E., Desmoulins, P. O., Chemin, K., Guia, S., Cremer, H., Ortaldo, J., Love, P., Kaiserlian, D. and Vivier, E. 2000. Combined natural killer cell and dendritic cell functional deficiency in KARAP/DAP12 loss-of-function mutant mice. Immunity 13:355.[ISI][Medline]
  16. Lanier, L. L., Corliss, B. C., Wu, J., Leong, C. and Phillips, J. H. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391:703.[CrossRef][ISI][Medline]
  17. Lanier, L. L., Corliss, B., Wu, J. and Phillips, J. H. 1998. Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 8:693.[ISI][Medline]
  18. Mason, L. H., Anderson, S. K., Yokoyama, W. M., Smith, H. R., Winkler-Pickett, R. and Ortaldo, J. R. 1996. The Ly-49D receptor activates murine natural killer cells. J. Exp. Med. 184:2119.[Abstract/Free Full Text]
  19. Diefenbach, A., Jamieson, A. M., Liu, S. D., Shastri, N. and Raulet, D. H. 2000. Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nat. Immunol. 1:119.[CrossRef][ISI][Medline]
  20. Djeu, J. Y., Jiang, K. and Wei, S. 2002. A view to a kill: signals triggering cytotoxicity. Clin. Cancer Res. 8:636.[Abstract/Free Full Text]
  21. Chuang, S. S., Kim, M. H., Johnson, L. A., Albertsson, P., Kitson, R. P., Nannmark, U., Goldfarb, R. H. and Mathew, P. A. 2000. 2B4 stimulation of YT cells induces natural killer cell cytolytic function and invasiveness. Immunology 100:378.[CrossRef][ISI][Medline]
  22. Ogasawara, K., Yoshinaga, S. K. and Lanier, L. L. 2002. Inducible costimulator costimulates cytotoxic activity and IFN-gamma production in activated murine NK cells. J. Immunol. 169:3676.[Abstract/Free Full Text]
  23. Colucci, F., Schweighoffer, E., Tomasello, E., Turner, M., Ortaldo, J. R., Vivier, E., Tybulewicz, V. L. and Di Santo, J. P. 2002. Natural cytotoxicity uncoupled from the Syk and ZAP-70 intracellular kinases. Nat. Immunol. 3:288.[CrossRef][ISI][Medline]
  24. Jamieson, A. M., Diefenbach, A., McMahon, C. W., Xiong, N., Carlyle, J. R. and Raulet, D. H. 2002. The role of the NKG2D immunoreceptor in immune cell activation and natural killing. Immunity 17:19.[ISI][Medline]
  25. Shi, F. D., Wang, H. B., Li, H., Hong, S., Taniguchi, M., Link, H., Van Kaer, L. and Ljunggren, H. G. 2000. Natural killer cells determine the outcome of B cell-mediated autoimmunity. Nat. Immunol. 1:245.[CrossRef][ISI][Medline]
  26. Matsumoto, Y., Kohyama, K., Aikawa, Y., Shin, T., Kawazoe, Y., Suzuki, Y. and Tanuma, N. 1998. Role of natural killer cells and TCR gamma delta T cells in acute autoimmune encephalomyelitis. Eur. J. Immunol. 28:1681.[CrossRef][ISI][Medline]
  27. Fort, M. M., Leach, M. W. and Rennick, D. M. 1998. A role for NK cells as regulators of CD4+ T cells in a transfer model of colitis. J. Immunol. 161:3256.[Abstract/Free Full Text]
  28. Adorini, L., Gregori, S. and Harrison, L. C. 2002. Understanding autoimmune diabetes: insights from mouse models. Trends Immunol. 8:31.
  29. Kataoka, S., Satoh, J., Fujiya, H., Toyota, T., Suzuki, R., Itoh, K. and Kumagai, K. 1983. Immunologic aspects of the nonobese diabetic (NOD) mouse. Abnormalities of cellular immunity. Diabetes 32:247.[Abstract]
  30. Poulton, L. D., Smyth, M. J., Hawke, C. G., Silveira, P., Shepherd, D., Naidenko, O. V., Godfrey, D. I. and Baxter, A. G. 2001. Cytometric and functional analyses of NK and NKT cell deficiencies in NOD mice. Int. Immunol. 13:887.[Abstract/Free Full Text]
  31. Carnaud, C., Gombert, J., Donnars, O., Garchon, H. and Herbelin, A. 2001. Protection against diabetes and improved NK/NKT cell performance in NOD.NK1.1 mice congenic at the NK complex. J. Immunol. 166:2404.[Abstract/Free Full Text]
  32. Shultz, L. D., Schweitzer, P. A., Christianson, S. W., Gott, B., Schweitzer, I. B., Tennent, B., McKenna, S., Mobraaten, L., Rajan, T. V., Greiner, D. L., et al. 1995. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J. Immunol. 154:180.[Abstract/Free Full Text]
  33. Ogasawara, K., Hamerman, J. A., Hsin, H., Chikuma, S., Bour-Jordan, H., Chen, T., Pertel, T., Carnaud, C., Bluestone, J. A. and Lanier, L. L. 2003. Impairment of NK cell function by NKG2D modulation in NOD mice. Immunity 18:41.[ISI][Medline]
  34. Arase, H., Saito, T., Phillips, J. H. and Lanier, L. L. 2001. Cutting edge: the mouse NK cell-associated antigen recognized by DX5 monoclonal antibody is CD49b (alpha 2 integrin, very late antigen-2). J. Immunol. 167:1141.[Abstract/Free Full Text]
  35. Tanaka, T., Tsudo, M., Karasuyama, H., Kitamura, F., Kono, T., Hatakeyama, M., Taniguchi, T. and Miyasaka, M. 1991. A novel monoclonal antibody against murine IL-2 receptor beta-chain. Characterization of receptor expression in normal lymphoid cells and EL-4 cells. J. Immunol. 147:2222.[Abstract/Free Full Text]
  36. Wicker, L. S., Todd, J. A. and Peterson, L. B. 1995. Genetic control of autoimmune diabetes in the NOD mouse. Annu. Rev. Immunol. 13:179.[CrossRef][ISI][Medline]
  37. Vyse, T. J. and Todd, J. A. 1996. Genetic analysis of autoimmune disease. Cell 85:311.[ISI][Medline]
  38. Gidlund, M., Orn, A., Wigzell, H., Senik, A. and Gresser, I. 1978. Enhanced NK cell activity in mice injected with interferon and interferon inducers. Nature 273:759.[ISI][Medline]
  39. Kim, S., Iizuka, K., Kang, H.-S. P., Dokun, A., French, A. R., Greco, S. and Yokoyama, W. M. 2002. In vivo developmental stages in murine natural killer cell maturation. Nat. Immunol. 3:523.[CrossRef][ISI][Medline]
  40. Idris, A. H., Smith, H. R., Mason, L. H., Ortaldo, J. R., Scalzo, A. A. and Yokoyama, W. M. 1999. The natural killer gene complex genetic locus Chok encodes Ly-49D, a target recognition receptor that activates natural killing. Proc. Natl Acad. Sci. USA 96:6330.[Abstract/Free Full Text]
  41. Idris, A. H., Iizuka, K., Smith, H. R., Scalzo, A. A. and Yokoyama, W. M. 1998. Genetic control of natural killing and in vivo tumor elimination by the Chok locus. J. Exp. Med. 188:2243.[Abstract/Free Full Text]
  42. Nakamura, M. C., Naper, C., Niemi, E. C., Spusta, S. C., Rolstad, B., Butcher, G. W., Seaman, W. E. and Ryan, J. C. 1999. Natural killing of xenogeneic cells mediated by the mouse Ly-49D receptor. J. Immunol. 163:4694.[Abstract/Free Full Text]
  43. Höglund, P., Öhlën, C., Carbone, E., Franksson, L., Ljunggren, H. G., Latour, A., Koller, B. and Kärre, K. 1991. Recognition of beta 2-microglobulin-negative (beta 2m) T-cell blasts by natural killer cells from normal but not from beta 2m mice: nonresponsiveness controlled by beta 2m bone marrow in chimeric mice. Proc. Natl Acad. Sci. USA 88:10332.[Abstract]
  44. Liao, N. S., Bix, M., Zijlstra, M., Jaenisch, R. and Raulet, D. 1991. MHC class I deficiency: susceptibility to natural killer (NK) cells and impaired NK activity. Science 253:199.[ISI][Medline]
  45. Lyons, A. B. and Parish, C. R. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171:131.[CrossRef][ISI][Medline]
  46. Oehen, S., Brduscha-Riem, K., Oxenius, A. and Odermatt, B. 1997. A simple method for evaluating the rejection of grafted spleen cells by flow cytometry and tracing adoptively transferred cells by light microscopy. J. Immunol. Methods 207:33.[CrossRef][ISI][Medline]
  47. Höglund, P., Glas, R., Öhlën, C., Ljunggren, H. G. and Kärre, K. 1991. Alteration of the natural killer repertoire in H-2 transgenic mice: specificity of rapid lymphoma cell clearance determined by the H-2 phenotype of the target. J. Exp. Med. 174:327.[Abstract]
  48. Lauwerys, B. R., Renauld, J.-C. and Houssiau, F. A. 1999. Synergistic proliferation and activation of Natural Killer cells by interleukin 12 and interleukin 18. Cytokine 11:822.[CrossRef][ISI][Medline]
  49. McVicar, D. W., Winkler-Pickett, R., Taylor, L. S., Makrigiannis, A., Bennett, M., Anderson, S. K. and Ortaldo, J. R. 2002. Aberrant DAP12 signaling in the 129 strain of mice: implications for the analysis of gene-targeted mice. J. Immunol. 169:1721.[Abstract/Free Full Text]
  50. Bakker, A. B., Hoek, R. M., Cerwenka, A., Blom, B., Lucian, L., McNeil, T., Murray, R., Phillips, L. H., Sedgwick, J. D. and Lanier, L. L. 2000. DAP12-deficient mice fail to develop autoimmunity due to impaired antigen priming. Immunity 13:345.[ISI][Medline]
  51. Lowin-Kropf, B., Kunz, B., Schneider, P. and Held, W. 2002. A role for the src family kinase Fyn in NK cell activation and the formation of the repertoire of Ly49 receptors. Eur. J. Immunol. 32:773.[CrossRef][ISI][Medline]
  52. Chan, G., Hanke, T. and Fischer, K. D. 2001. Vav-1 regulates NK T cell development and NK cell cytotoxicity. Eur. J. Immunol. 31:2403.[CrossRef][ISI][Medline]
  53. Diefenbach, A., Tomasello, E., Lucas, M., Jamieson, A. M., Hsia, J. K., Vivier, E. and Raulet, D. H. 2002. Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D. Nat. Immunol. 3:1142.[CrossRef][ISI][Medline]
  54. Gilfillan, S., Ho, E. L., Cella, M., Yokoyama, W. M. and Colonna, M. 2002. NKG2D recruits two distinct adapters to trigger NK cell activation and costimulation. Nat. Immunol. 3:1150.[CrossRef][ISI][Medline]
  55. Lowin-Kropf, B., Kunz, B., Beermann, F. and Held, W. 2002. Impaired natural killing of MHC class I-deficient targets by NK cells expressing a catalytically inactive form of SHP-1. J. Immunol. 165:1314.
  56. Lowin-Kropf, B. and Held, W. 2000. Positive impact of inhibitory Ly49 receptor–MHC class I interaction on NK cell development. J. Immunol. 165:91.[Abstract/Free Full Text]
  57. Tsutsui, H., Nakanishi, K., Matsui, K., Higashino, K., Okamura, H., Miyazawa, Y., Kaneda, K. 1996. IFN-gamma-inducing factor upregulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones. J. Immunol. 157:3967.[Abstract]
  58. Hashimoto, W., Osaki, T., Okamura, H., Robbins, P. D., Kurimoto, M., Nagata, S., Lotze, M. T. and Tahara, H. 1999. Differential antitumor effects of administration of recombinant IL-18 or recombinant IL-12 are mediated primarily by Fas–Fas ligand- and perforin-induced tumor apoptosis respectively. J. Immunol. 163:583.[Abstract/Free Full Text]
  59. Screpanti. V., Wallin, R. P., Ljunggren, H.-G. and Grandien, A. 2001. A central role for death receptor-mediated apoptosis in the rejection of tumors by NK cells. J. Immunol. 167:2068.[Abstract/Free Full Text]
  60. Sato, K., Hida, S., Takayanagi, H., Yokochi, T., Nobuhiko, K., Takeda, K., Yagita, H., Okomura, K., Tanaka, N., Taniguchi, T. and Ogasawara, K. 2001. Antiviral response by natural killer cells through TRAIL gene induction by IFA-{alpha}/ß. Eur. J. Immunol. 31:3138.[CrossRef][ISI][Medline]
  61. Algarra, I., Perez, M., Hoglund, P., Gaforio, J. J., Ljunggren, H. G. and Garrido, F. 1993. Generation and control of metastasis in experimental tumor systems; inhibition of experimental metastases by a tilorone analogue. Int. J. Cancer 54:518.[ISI][Medline]
  62. Afifi, M. S., Kumar, V. and Bennett, M. 1985. Stimulation of genetic resistance to marrow grafts in mice by interferon-alpha/beta. J. Immunol. 134:3739.[Abstract/Free Full Text]
  63. Yu, Y. Y., Kumar, V. and Bennett, M. 1992. Murine natural killer cells and marrow graft rejection. Annu. Rev. Immunol. 10:189.[CrossRef][ISI][Medline]
  64. Serreze, D. V., Hamaguchi, K. and Leiter, E. H. 1989. Immunostimulation circumvents diabetes in NOD/Lt mice. J. Autoimmun. 2:759.[ISI][Medline]
  65. Rothe, H., Hausmann, A., Casteels, K., Okamura, H., Kurimoto, M., Burkart, V., Mathieu, C. and Kolb, H. 1999. IL-18 inhibits diabetes development in nonobese diabetic mice by counterregulation of Th1-dependent destructive imsulitis. J. Immunol. 163:1230.[Abstract/Free Full Text]