Natural killing of MHC class I lymphoblasts by NK cells from long-term bone marrow culture requires effector cell expression of Ly49 receptors
Jakob Lindberg,
Alfonso Martín-Fontecha and
Petter Höglund
Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Box 280, 17177 Stockholm, Sweden
Correspondence to:
P. Höglund
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Abstract
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NK cells from long-term bone marrow culture (LTBMC) were compared with IL-2-activated splenic NK cells [short-term spleen cell culture (STSC)] with regard to expression of inhibitory Ly49 receptors and cytotoxic function. In the LTBMC, the total number of NK cells expressing either one of the Ly49 molecules A, C/I and G2 was strongly reduced (1015% of NK1.1+ cells) compared to the STSC (8090% of NK1.1+ cells). With regard to cytotoxic function, we confirmed that LTBMC-derived NK cells efficiently killed the prototype NK target YAC-1. However, against other targets, killing was more variable. First, while STSC-derived NK cells clearly distinguished MHC class I from MHC class I+ tumor cell targets, LTBMC-derived NK cells did not; they either killed both targets equally well or not at all. Secondly, LTBMC-derived NK cells were largely incapable of killing lymphoblast targets deficient in MHC class I expression. To test whether this cytotoxic defect was due to the low number of Ly49+ NK cells in the LTBMC, we separated Ly49+ and Ly49 NK cells by cell sorting and tested them individually. This experiment showed that only Ly49+ NK cells in the LTBMC were able to kill MHC class I lymphoblasts (and to distinguish them from MHC class I+), despite good cytotoxicity against YAC-1 cells in both populations. These data suggest that certain modes of NK cell triggering are dependent on Ly49 receptor expression. From our results, we speculate that inhibitory receptors are expressed before triggering receptors for normal self cells during NK cell development, which may be an important mechanism to preserve self tolerance during the early stages of NK cell maturation.
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Introduction
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NK cell cytotoxicity is regulated by triggering as well as inhibitory signals (1). A two-step model suggests that initial activation of NK cell cytotoxicity is modulated by down-regulating signals, resulting from binding of inhibitory receptors on NK cells to MHC class I molecules on target cells (2). Thus, triggering proceeds as a default pathway unless the dominant negative signal is elicited (3,4), which implies the negative signals as critical to avoid autoreactivity. The positive signals that trigger NK cell cytotoxicity in defined biological situations are poorly characterized, despite the fact that several activating receptors have been identified (reviewed in 5). In contrast, the inhibitory pathways, in the mouse mediated by lectin-like Ly49 and CD94/NKG2 receptors, are better known. The Ly49 receptors, which interact with various alleles of classical MHC class I molecules, are expressed by subsets of NK cells in a complex, as yet not fully understood, pattern (2). In addition to Ly49 receptors, many NK cells also express CD94/NKG2, an inhibitory receptor that interacts with the non-classical MHC class I molecule Qa-1 complexed with peptides derived from classical MHC class I molecules (6,7). The genes for these inhibitory receptors are all located in the NK complex on mouse chromosome six (8). At least nine Ly49 and two NKG2 genes are known. In contrast, CD94 appears to be encoded by a single gene.
It has been proposed that self tolerance in mature NK cells depend on expression of Ly49 receptors. If every NK cell expresses at least one receptor for self MHC class I, they will be unable to kill normal self cells (9). The finding that NK cells can kill normal self cells if the relevant Ly49 receptors on the NK cells are blocked with antibodies is in line with this hypothesis (1012). Similarly, MHC class I disparate non-self cells (i.e. allogeneic or MHC class I lymphoblasts) are killed by NK cells because they lack some self MHC class I molecules (3,4) and Ly49 receptors on all NK cells are therefore not engaged. Thus, NK cells express triggering receptors for normal self cells that are allowed to dominate when the interaction between inhibitory receptors and MHC class I is disrupted.
An important question is how the balance between positive and negative signals is tuned during NK cell development to avoid autoreactivity in the maturing NK cell population. For example, how is expression of inhibitory receptors regulated in time and how does this relate to the capacity of NK cells to kill different target cells? It was recently shown that Ly49 expression is absent from birth and that the numbers of Ly49+ NK cells in the spleen reach adult levels only after several weeks (13,14). Interestingly, the kinetics of Ly49 receptor acquisition parallels the development of certain NK cell manifestations, such as the ability to kill YAC-1 cells in vitro (15) and to mediate rejection of MHC incompatible bone marrow in vivo (16). This association suggests that in order for an NK cell to acquire full effector functions, expression of Ly49 receptors is required. However, direct evidence for an association between NK cell function and Ly49 receptor expression has not been demonstrated.
To study this, an in vitro system that would mimic normal in vivo differentiation of NK cells, including the expression of Ly49 receptors, would be useful. We evaluated the previously described long-term bone marrow culture (LTBMC) system in this respect (1719). In the LTBMC, bone marrow cells are first cultured without addition of growth factors for 4 weeks. During this period cellularity drops to 12% of the input number and only adherent stromal cells surrounded by few non-adherent round cells are observed. At this stage, stimulation with IL-2 for 2 weeks leads to a large expansion of cells in the culture, which in the end results in a virtually pure population of NK1.1+ cells (18). Previous experiments have shown that such LTBMC-derived NK cells share many properties of mature NK cells, including high YAC-1 killing ability (1719). In this report, we compared LTBMC-derived NK cells with NK cells derived from a short-term spleen culture (STSC) with regard to cytotoxic capacity and the expression of inhibitory Ly49 receptors. We conclude that the NK cells generated in these two ways are severely different. While most STSC-derived NK cells expressed Ly49 receptors, LTBMC-derived NK cells lacked to a large extent, but not completely, Ly49 receptor expression. Furthermore, LTBMC-derived NK cells differed from STSC-derived NK cells in that they failed to kill MHC class I lymphoblasts, and could not distinguish between MHC class I and MHC class I+ tumor target cells. Interestingly, the triggering defect against MHC class I lymphoblasts was confined to the Ly49 NK cell population: the few Ly49+ NK cells present in the LTBMC displayed a normal pattern of cytotoxicity. These results show for the first time that acquisition of Ly49 receptors is associated with the capacity to kill certain normal target cells. We hypothesize that this link is important to avoid NK cell killing of self cells during some stages of NK cell development.
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Methods
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Tumor cell lines
Tumor cell lines were cultured in RPMI 1640 supplemented with 5% FCS (v/v). YAC-1 is a Moloney murine leukemia virus-induced T cell lymphoma of the A/Sn strain. RMA is a subline of the Raucher virus-induced T cell lymphoma RBL-5 (H-2b) and RMA-S is a TAP2 variant of RMA. B16 is a B6-derived melanoma cell line (H-2b) and P815 is a mastocytoma cell line derived from the DBA/2 strain (H-2d).
Generation of NK cells in LTBMC and STSC
To generate LTBMC cells, bone marrow was collected from femurs of B6 mice (by flushing the bone marrow cavity with PBS) and cultured at 2.5x106 cells/ml for 4 weeks in RPMI 1640 medium supplemented with 5% FCS, L-glutamine, penicillin, streptomycin and 2-mercaptoethanol. After 4 weeks, 1000 U/ml rIL-2 was added and 1315 days later experiments were performed. To generate STSC cells, erythrocyte-depleted spleen cells were cultured for 4 days at 2.5x106 cells/ml in
-MEM supplemented with 5% FCS, 10 mM HEPES, 2-mercaptoethanol, penicillin, streptomycin and 1000 U/ml rIL-2. Both LTBMC and STSC cultures were grown under 10% CO2 conditions.
Generation of concanavalin A (Con A)-activated T cell blasts and in vitro cytotoxicity assay
Spleen cells were depleted from erythrocytes and incubated for 48 h (37°C) at 2x106 cells/ml in RPMI 1640 medium supplemented with 5% FCS, penicillin, streptomycin and 3 µg/ml Con A (Sigma, St Louis, MO). After labeling of target cells with 51Cr (for 1 h at 37°C), cells were diluted to the proper concentration in medium. Each E:T ratio was assayed in triplicate and 2x1035x103 target cells were used per well depending on the experiment. After 4 h of incubation, 100 µl of supernatant was harvested and the radioactivity of each sample was determined by counting
radiation.
Flow cytometry and cell sorting
The following mAb were used: NK1.1 [PK136; phycoerythrin (PE)-conjugated], CD3 (145-2C11; FITC-conjugated), B220 (RA3-6B2; PE-conjugated), CD69 (H1.2F3; FITC-conjugated), Ly49A (A1; biotinylated), Ly49C/I (5E6; biotinylated) and Ly49G2 (4D11; FITC-conjugated). The anti-Ly49G2-producing hybridoma (4D11) was originally acquired from ATCC (Rockville, MD). All other antibodies were purchased from PharMingen (San Diego, CA). To stain cells for flow cytometry, cells from LTBMC and STSC were washed once with PBS, incubated with directly labeled specific antibody for 3060 min on ice and washed again prior to analysis. To sort Ly49+ and Ly49 NK cells, LTBMC-derived cells were stained with antibodies against the inhibitory Ly49 molecules A, C/I and G2 (directly FITC-conjugated antibodies or biotinylated antibodies followed by streptavidin-FITC) together with NK1.1PE. Sorted cells were replated in fresh medium with IL-2 and were allowed to recover in the incubator overnight before testing for killing ability against lymphoblasts the next day. Analytical flow cytometry was performed using a FACScan (Becton Dickinson, Mountain View, CA). Dead cells were excluded by means of an electronic gate in the forward and side scatter profile. After acquisition, the FACS profiles were analyzed using CellQuest software. Cell sorting experiments were performed using a FACS Vantage (Becton Dickinson).
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Results
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IL-2-activated LTBMC-derived cells show the phenotype of activated NK cells but are deficient in Ly49 receptor expression
After culture of 25x106 bone marrow cells for 4 weeks, only a few round cells and some stromal-like cells were left in the flask. Stimulation with IL-2 resulted in extensive proliferation in the culture; the stromal-like cells disappeared, and the cell population became homogenous and dominated by round blast-like cells. 1315 days after IL-2 stimulation, 7590% of the cells in the LTBMC were NK1.1+, CD3, B220 and CD69+ (Fig. 1
). LTBMC-derived NK cells were therefore classified as activated NK cells. With the exception of CD69 staining, that was not previously analyzed, this result was noted also in previous studies (1719). A large difference was found in the expression of Ly49 receptors. In the experiment shown in this study, 25% of NK1.1+ cells were Ly49A+, 31% were Ly49C/I+ and 59% were Ly49G2+ in the STSC, while among LTMBC-derived NK cells, 4% were Ly49A+, 1% were Ly49C/I+ and 5% were Ly49G2+ (Fig. 2
). Table 1
summarizes the results from several experiments.

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Fig. 1. FACS analysis of LTBMC- and STSC-derived cells. The histograms represent single-color stainings except for CD69, which is on gated NK1.1+ cells. The dotted line in the CD69 histograms represents background staining without antibody.
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Fig. 2. Expression of Ly49 receptors on LTBMC- and STSC-derived NK cells from B6 mice. Histograms show expression of the NK cell receptors Ly49A, Ly49C/I and Ly49G2 on gated NK1.1+ cells.
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LTBMC-derived NK cells kill tumor cells and ß2-microglobulin (ß2m) lymphoblasts with variable efficiency
When LTBMC- and STSC-derived NK cells were compared in cytotoxicity assays, a complex situation was revealed. As was already reported (1719), LTBMC-derived NK cells were always efficient in killing the prototype NK cell target YAC-1. However, against other targets, the cytotoxic potency of LTBMC-derived NK cells was more unpredictable than that of STSC-derived NK cells. In some experiments, LTBMC-derived NK cells were as efficient as those from STSC in tumor cell killing, both against hematopoietic tumors and tumors originating from other tissues (Fig. 3
). However, in other experiments, morphologically normal LTBMC cultures displayed very poor killing ability. Table 2
lists nine consecutive experiments in which LTBMC- and STSC-derived NK cells were compared with respect to killing capacity against three tumor cell lines: YAC-1, RMA and RMA-S, and against two normal target cells, ß2m+ and ß2m Con A blasts. With regard to the tumor targets, STSC-derived NK cells rarely failed to kill any of them (Table 2
). In contrast, despite being always cytotoxic against YAC-1 cells, LTBMC-derived NK cells sometimes completely failed to kill the RMA and RMA-S tumors (Table 2
, experiments 2, 4 and 7), but sometimes killed them well (Table 2
, experiments 1, 3 and 8). Interestingly, in the cases where RMA and RMA-S target cells were killed, we were surprised to find no difference in sensitivity between the two target cells, which was in striking contrast to STSC-derived NK cells, that almost always killed the MHC class I RMA-S cells better than MHC class I+ RMA (Table 2
).

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Fig. 3. In vitro cytotoxicity assay using STSC cells and LTBMC cells from B6 mice against tumor cell lines YAC-1, RMA, RMA-S, B16 and P815.
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LTBMC-derived NK cells were also tested against ß2m lymphoblasts. Here, a different pattern appeared. In contrast to the clear capacity (although variable) to kill both RMA and RMA-S targets, LTBMC-derived NK cells were more consistently unable to kill both ß>2m and ß2m+ lymphoblasts (Table 2
). This was again different from STSC-derived NK cells, that always killed ß2m lymphoblasts better than ß2m+ (Table 2
). However, LTBMC-derived NK cells did occasionally manage to kill the former targets (i.e. Table 2
, experiment 8), but the lack of killing against ß2m targets was the most commonly observed result (Table 1
, experiments 2, 5, 6, 7 and 9). It should be noted that although the level of YAC-1 lysis was in the lower range (<40%) in many of the experiments in which LTBMC-derived NK cells failed to distinguish between ß>2m and ß2m+ lymphoblasts, there were exceptions to this (experiment 6, Table 2
). Thus, we believe that the lack of distinction between ß2m and ß2m+ lymphoblasts reflects a qualitative, rather than quantitative, defect in the LTBMC-derived NK cell population compared to STSC-derived NK cells. This notion is supported by two other observations. First, STSC-derived NK cells always distinguished ß2m from ß2m+ lymphoblasts despite low YAC-1 killing capacity (i.e. experiment 1, Table 2
). Secondly, sorted Ly49+ LTBMC-derived NK cells clearly distinguished ß2m and ß2m+ lymphoblasts despite low YAC-1 killing (Table 3
, experiment 1 and 3, see next section).
Ly49+, but not Ly49, LTBMC-derived NK cells are able to kill ß2m lymphoblasts.
We wondered whether the differences in target cell specificity of LTBMC-derived NK cells relative to STSC-derived was related to the Ly49 deficiency in the LTBMC. In particular, we were intrigued by the inability of LTBMC-derived NK cells to kill ß2m lymphoblasts, something that STSC-derived NK cells never failed to do. To investigate this directly, we separated Ly49+ and Ly49 LTBMC-derived NK cells by cell sorting (Fig. 4
), and tested the two populations separately for ability to lyse ß2m lymphoblasts. Strikingly, when Ly49+ NK cells were enriched and tested separately, they displayed completely normal killing of ß2m lymphoblasts (Table 3
). In contrast, Ly49 NK cells were always unable to kill ß2m lymphoblasts, despite good killing against YAC-1 cells (Table 3
). This result showed that Ly49 receptor expression was associated with the ability to kill normal self cells and also suggested that the occasional discriminatory capacity of unsorted LTBMC-derived NK cells we observed (especially pronounced in experiment 3, Table 3
, but also seen in experiment 2, Table 3
and experiment 8, Table 2
) could be due to an unusually high percentage, or activity, of Ly49+ NK cells in those cultures.

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Fig. 4. Sorting of Ly49+ and Ly49 populations. Cells from LTBMC were double stained with antibody against NK1.1 (FL2) and a pool of antibodies against Ly49A, C, I and G2 (FL1), and sorted. The dot-plot to the left shows the sample before sorting and the histograms to the right show purity of the populations directly after sorting.
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Discussion
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We report that, in our hands, the LTBMC system failed to give rise to mature NK cells with similar characteristics as NK cells generated from the spleen. We noted some similarities, but also some interesting differences between these two NK cell populations, both with regard to function and to expression of cell surface markers.
Phenotypic profiles
LTBMC- and STSC-derived NK cells were both positive for cell surface antigens characteristic for activated NK cells, such as the NKR-P1 allele NK1.1 and the early activation antigen CD69. In agreement with earlier reports investigating LTBMC-derived NK cells, we found that most of the LTBMC-derived NK cells were negative for B220, a high molecular isoform of the CD45 antigen. B220 has been suggested as a marker for cytokine-activated NK cells with lytic ability (20) and an interesting possibility is therefore that the lack of B220 is somehow related to the cytotoxic deficiency of LTBMC-derived NK cells. However, the role of B220 in NK cell cytotoxicity is uncertain, given that NK cells in CD45 mice, lacking B220, have normal cytotoxicity (21). The functional significance of the lack of B220 expression in the LTBMC therefore requires further study.
Another phenotypic difference was the profound deficiency in expression of inhibitory Ly49 receptors seen in LTBMC-derived NK cells. Previous results from other in vitro systems also demonstrated deficient Ly49 receptor expression on NK cells generated from immature precursors (2225). One explanation as to why full differentiation of NK cells is not seen in these systems could be that that the final differentiation of effector NK cells leading to Ly49 expression requires a special combination of growth factors that is difficult to replicate satisfactorily in vitro. Relevant to this notion, we have tried to induce Ly49 expression in all NK cells in the LTBMC by adding different cytokines during the IL-2 induction phase (including IL-15), but without success (data not shown). It is also possible that the in vitro culture systems used fail to reproduce the correct microenvironment present in vivo that, in combination with the right growth factors, would be needed to ensure proper contacts between potentially selecting stromal cells and the NK cell precursor during differentiation. However, not all NK cells in the LTBMC were Ly49. We consistently found 35% of the NK cells staining positive for either one of the three antibodies against inhibitory Ly49 molecules we tested, directed against Ly49A, Ly49C/I and Ly49G2. In the spleen, those three antibodies in combination stained ~8090% of all NK cells, but in the LTBMC, the total number of cells expressing any of those Ly49 molecules varied between 10 and 15%. Given the normal cytotoxic capacity of those NK cells (see below), they probably represent fully mature NK cells, similar to those found in the spleen. However, it is unlikely that those NK cells originate from contaminating mature cells from the original bone marrow isolate, because before addition of IL-2, the bone marrow is left untouched in the culture flask for 4 weeks, a step which should eliminate any fully differentiated mature NK cells present in the culture (19). In addition, no cells expressing NK1.1 could be detected in the culture after 4 weeks of starvation, suggesting that no mature NK cells were present at the start of the IL-2 culture (data not shown and 19).
Why are few, but not all, NK cells Ly49+ in the LTBMC? One possibility is that the initial step in NK cell development could be initiated in the NK cell precursor with a certain probability by an intrinsic developmental pathway, independent of exogenous growth factors. Alternatively, the correct maturation factors may be present in the LTBMC only at a suboptimal level, which, due to competition for limited resources, is sufficient to trigger maturation of few, but not all, NK cells. A third possibility is that the Ly49 LTBMC-derived NK cells represent an expanded sub-population of normal fully differentiated NK cells that do not express inhibitory receptors and whose survival is particularly favored by the LTBMC culture conditions. At present, we cannot distinguish between these alternatives.
Cytotoxic function
Two intriguing observations were made with regard to the cytotoxic function of LTBMC-derived NK cells. The first was that they were unable to distinguish between RMA and RMA-S tumor cells, despite the fact that RMA-S cells are severely deficient in MHC class I expression and that RMA express high levels (26). This result was surprising in light of recent data showing that IL-2-activated splenic NK cells from neonatal mice (<10 days) were clearly able, despite Ly49 deficiency, to distinguish between self cells with high MHC class I expression from those with low expression, including the RMA/RMA-S target cell pair (13,14). One possible difference between neonatal and LTBMC-derived NK cells could be that the former, but not the latter, express inhibitory receptors different from Ly49, candidates of which are CD94/NKG2 receptors (6,7,27,28). This hypothesis would explain why neonatal, but not LTBMC-derived, NK cells can distinguish RMA from RMA-S cells. How would such a difference come about? In this respect, it may be important that the LTBMC protocol starts with a 4 week resting period of the input bone marrow, during which there is excessive cell death and by this selective enrichment of multipotent hematopoietic bone marrow-derived stem cells (1719). In the other culture systems, the starting material is obtained either from outside of the bone marrow or by direct sorting of previously identified committed progenitors from bone marrow, which in both cases may lead to expansion of stem cells from later stages in NK cell development (2225). Future studies will be needed to carefully compare expression of inhibitory receptors and cytotoxic function on NK cells derived from the various culture systems that exist.
The second observation was that Ly49 LTBMC-derived NK cells were incapable of killing non-transformed ß2m cells despite the fact that they were cytotoxic against YAC-1 cells. Since ß2m lymphoblasts are normally very good targets for activated NK cells (3,4), this result demonstrated that Ly49 and Ly49+ LTBMC-derived NK cells were qualitatively different in some aspects of target cell specificity, and that there exists a link between the ability to be triggered by normal self cells and expression of inhibitory Ly49 receptors. This is an interesting result in relation to self tolerance development. It has been suggested that NK cell tolerance depends upon NK cell expression of allele-specific Ly49 receptors (9,29). This theory is based on the notion that NK cells normally possess activating pathways triggered by self cells. If these activating pathways would be triggered in the absence of inhibitory receptors, a risk for autoimmune cytotoxicity would prevail. It therefore makes sense that lack of inhibitory receptors would preclude expression of triggering pathways activated by normal self cells. Also in this case, the comparison with Ly49 neonatal NK cells, which were capable of not only distinguishing RMA from RMA-S cells but also to kill MHC class I lymphoblasts, is interesting. Perhaps the inhibitory receptors different from Ly49 that are expressed by neonatal NK cells are sufficient to signal that self tolerance is secured. This in turn would allow the NK cell to go through the differentiation program regulating expression of a full set of activating receptors. For reasons unknown at this point, this checkpoint would not be passed in the LTBMC and LTBMC-derived NK cells would therefore be prevented to express triggering receptors for self cells.
Finally, Ly49 NK cells were clearly capable of killing tumor cells despite the absence of lymphoblast killing. How can this be explained? There are several lines of evidence to suggest that tumor cells and lymphoblasts are recognized differently (30). For example, NK cells in mice lacking the CD4 homologue LAG3 showed impaired tumor recognition in vitro, but were perfectly capable of mediating rejection of ß2m bone marrow grafts in vivo (31). Another example is self tolerance in ß2m mice. ß2m NK cells have completely lost the capacity to reject ß2m bone marrow grafts and to kill ß2m Con A lymphoblast targets, while the ability to be triggered by tumor-specific activating ligands is largely unaffected (32). Thus, NK cells may use, at least in part, different recognition systems in the killing of lymphoblasts versus tumor target cells. Our data suggest that the function of those triggering structures, be it specific receptors or different intracellular signaling pathways, are regulated independently, such that one can be allowed to develop normally separated from each other.
In summary, we have reported a correlation between expression of Ly49 receptors and cytotoxic function; in LTBMC-derived NK cells, expression of Ly49 receptors was necessary to allow killing of lymphoblasts deficient in MHC class I expression. We suggest that this correlation reflects a developmentally ordered expression of triggering versus inhibitory MHC class I-specific receptors during development. In this model, the inhibitory receptors must be expressed first to avoid autoreactivity during NK cell maturation.
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Acknowledgments
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We thank Klas Kärre for support and valuable discussions, Mats Olsson-Alheim for providing FITC-conjugated Ly49G2 antibody, Maria H. Johansson for comments on this manuscript, and Ros-Mari Johansson and her staff for excellent animal husbandry. This study was supported from the Swedish Cancer Society, the Karolinska Institute and the Åke Wiberg Foundation. A. M. F. was supported by the Swedish Cancer Society.
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Abbreviations
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ß2m | ß2-microglobulin |
Con A | concanavalin A |
LTBMC | long-term bone marrow culture |
PE | phycoerythrin |
STSC | short-term spleen cell culture |
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Notes
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Transmitting editor: J. F. Bach
Received 10 June 1998,
accepted 20 April 1999.
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