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Department of Clinical and Experimental Medicine, Section of Pharmacology, University of Perugia, 06100 Perugia, Italy [D. V. D., B. D. M., E. A., G. N., S. B., C. R.]; Karolinska Institute MTC, Stockholm, Sweden [M. S., H-G. L.]; and Department Scienza del Farmaco, University G. D’Annunzio, 66100 Chieti, Italy [L. B.]
Abstract
To investigate the role of MHC class I on in vitro differentiation of natural killer (NK) cells, a CD44low/-CD2-classIlow population was isolated from mouse bone marrow. This population, which lacked expression of NK-1.1, Ly49A, Ly49C/I, and Ly49G, generated populations of NK-1.1+ NK cells expressing Ly49A, Ly49C/I, or Ly49G when cocultured for 13 days with syngeneic supportive stromal cells in the presence of interleukin 2. Ly49A and Ly49C/I were absent on the progeny of progenitors tested after 7 days of culture but were expressed as a late event together with low-level expression of NK-1.1, from day 8 of culture. The addition of anti-H-2b monoclonal antibody to cultures at day 0 inhibited proliferation of progenitors supported by either syngeneic, allogeneic, or H-2b-deficient stromal cells, thus suggesting that the effect was not exerted on stromal cells. Additional analyses demonstrated that class Ilow progenitors generated class I+ cells on which the anti-H-2b monoclonal antibody exerted its inhibitory effect.
Introduction
The discovery of Ly49 family molecules in mouse and killer inhibitory receptors in humans has highlighted the importance of MHC class I recognition in both the killing of allogeneic and altered self cells and tolerance toward normal syngeneic cells by NK3 effector cells (1, 2, 3, 4) . It is not known in detail how and when NK cells acquire a repertoire of Ly49 molecules, allowing them to discriminate among different target cells. Specifically, the stage at which repertoire is acquired during the differentiation process from progenitors to mature NK cells and whether MHC class I molecules play a role in this process, as has been shown in thymic differentiation of CD8+ T cells, are not known.
Until now, the development of the NK repertoire has largely been limited to in vivo studies. In the course of these studies, Sykes et al. (5) found that expression of Ly49 molecules on NK cells is determined by interaction with class I molecules expressed on BM-derived radiosensitive cells and, to a lesser extent, by class I antigens expressed on radioresistant host elements. The importance of class I molecules in the creation of the NK repertoire was also highlighted by studies of mutant mice lacking ß2-microglobulin or the transporter protein Tap-1. Tap-1-/- mice, which express class I molecules at very low levels, generate a normal number of NK cells with an altered repertoire as compared with their littermates expressing normal levels of class I antigens (6) . Interestingly, in the same Tap-1-/- mice, the absence of class I antigens determines an almost complete lack of CD8+ T cells, indicating that the development of both CD8+ T and NK cells is influenced by class I molecules, albeit through different mechanisms. Of particular interest, in the work of Johansson et al. (7) , there was no evidence of deletion of potentially autoreactive NK cells, and NK cell tolerance development seemed rather to be the result of an adaptation process (7 , 8) due to the presence of cells in the environment in which differentiation of NK cells occurs. However, in these in vivo studies, it was not possible to assess whether the influence of class I molecules and Ly49 expression is exerted at a specific stage of the differentiation pathway of NK cells, e.g., on NK cell progenitors, or on their mature progeny. Dissection of these issues would greatly benefit from isolation of NK progenitors able to give rise to a progeny expressing Ly49s in in vitro cultures, and a recent report indicates that stromal cells are critical for the expression of Ly49 molecules (9) in differentiation of progenitors from adult BM, whereas in fetal NK cells, expression of Ly49E has been detected only at the mRNA level (10) .
In the present study, we isolated a CD44low/-CD2-class Ilow BM population from B6 mice (H-2b haplotype) that generated Ly49A-, Ly49C/I-, or Ly49G-positive and -negative NK cells after culture with IL-2 and irradiated BM stromal cells. The expression of Ly49 molecules was a late event in the differentiation process, and treatment with anti-H-2b class I mAb inhibited the ability of the progenitor population to generate NK cells by acting on progenitors.
Results
Characterization of the NK Precursor Population.
In our previous work, a murine BM subpopulation enriched for NK
precursors was isolated by a cell sorter and characterized as
CD44low/-TCR-NK-1.1-.
After stimulation with IL-2 and cocultivation on a discontinuous layer
of supportive stromal cells for 2 weeks, cells in this population were
able to give rise to a virtually pure
NK-1.1+TCR- cell
population with the ability to kill Yac-1 cells. The frequency of the
NK cell precursors was calculated by limiting dilution analysis to be
around 1:500 (11)
. To obtain a similar population by using
magnetic bead depletion, mAbs to CD44 and CD2 were used to obtain a
CD44low/-CD2-
subpopulation. Fig. 1
shows a flow cytometry analysis of BM cells before and after the
magnetic bead depletion procedure: Fig.1A
shows the isotypic
control for CD44 and CD2; Fig. 1B
shows unfractionated BM
stained for CD44 and CD2; and Fig. 1C
shows the BM after
depletion of CD44highCD2+
cells. Moreover, in Fig. 1, B and C
,
boxes formed by dashed lines indicate how we
discriminated CD44high from
CD44low/- cells, and the
box formed by solid lines indicates the
CD44low/-CD2- population.
Utilization of these markers allowed isolation of a NK cell
precursor-containing BM population with no contaminating mature NK
cells or other lineage-positive cells. This is because CD44 is
expressed at high intensity on most BM cells, including the majority of
T cells and all mature NK cells (11
, 12)
, and CD2 is also
expressed on T, NK, B, and pre-B cells (13
, 14)
. In
addition, MHC class I molecules, also expressed on mature NK
cells, was evaluated in the attempt to identify and enrich a precursor
population. Because a variable percentage of
CD44low/-CD2- cells were
class Ilow, they were depleted of the class
Ihigh cells (and were also Fc receptor negative
as determined using the 2.4G2 anti-Fc receptor mAb) for a subsequent
trial.
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95%)
should allow the complete elimination of any residual mature NK cells,
and this was confirmed by flow cytometry analysis. In Fig. 2, A–C
, flow cytometry stainings of unfractionated BM cells
with different NK cell markers are shown. In Fig. 2
, A—C,
the two-color staining shows the
NK-1.1+CD44high,
DX5+CD44high, or Ly49G
mature NK cells, respectively, present in the unfractionated BM. In
Fig. 2, D–F
, the flow cytometry analysis on the test
population obtained after BM depletion is shown.
NK-1.1+, DX5+, and
Ly49G+ mature NK cells completely disappeared
after the depletion steps (Fig. 1, D–F
, respectively).
However, as an additional test, we took advantage of a feature
discriminating NK cells derived from NK cell precursors and mature NK
cells. Mature NK cells proliferate after stimulation with IL-2 alone,
whereas precursors require not only IL-2 but also the presence of BM
stromal cells (9
, 11
, 12
, 16)
. In support of this
observation, a recent study (9)
showed that when a NK
progenitor population was deliberately contaminated with mature BM NK
cells, these latter cells were detected after stimulation with IL-2 in
the presence or absence of stroma, thus demonstrating that
proliferation of mature NK cells is not influenced by stromal cells. As
shown in Fig. 3
, no cells were detected at the end of the culture period when the
CD44low/-CD2-class
Ilow subpopulation was cultured with only IL-2,
without the support of stromal cells. When the same cells were cultured
with IL-2 on irradiated supportive stromal cells, the cell number
increased greatly starting from day 0 and reached a plateau from day 7
to day 13 (Fig. 3)
. These experiments strongly suggest that no mature
NK cells were present in the isolated BM subpopulation because they
were not detected even after stimulation with high doses of IL-2 that
would have induced their expansion.
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indicate that at day 3, almost all cells were negative for both markers
with very few (1.3 ± 0.2%)
NK-1.1lowCD3- cells. The
analysis at day 7 shows that two distinct cell populations were
generated: (a) a small
NK-1.1+CD3- population
(13 ± 11.3%); and (b) a larger
NK-1.1-CD3- population
(81.2 ± 12.4%). At day 13, virtually all cells (91.9 ±
3.4%) were NK-1.1+CD3-
cells. The fact that few contaminating CD3+ cells
[Fig. 3
, day 13 panel, 5.6 ± 2.4% of
CD3+NK-1.1+ cells
(top right quadrant) and 1.8 ± 1.2% of
CD3+NK-1.1- cells
(bottom right quadrant)] were present is a further indirect
demonstration that depletion of mature NK cells was effective. Because
T cells have a higher IL-2-dependent proliferation rate in the
unfractionated BM as compared with mature NK cells due to the
expression of high-affinity IL-2 receptor, are about double in number
(8–10% versus 3–4%, respectively), in the unfractionated
BM and are only about 7% at day 13 after the depletion steps, residual
mature NK cells will almost certainly be absent or, if present, will be
much less than the residual 7% T cells and thus will not substantially
contribute to the final NK cell number. These results suggest
that the
CD44low/-CD2-class
Ilow BM population contains NK cell precursors
that gave rise (day 13 dot plot) to a virtually pure population of NK
cells.
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, 10–15% of the NK cells were Ly49A+, 40–50%
were Ly49C/I+, and 48 ± 5.6% were
Ly49G+. To study at which point during the
differentiation pathway Ly49 molecules were expressed, the two cell
populations present at day 7 were analyzed. Fig. 6
shows that day 7 NK-1.1+ cells express Ly49A
(middle panel; top right quadrant) and Ly49C/I (bottom
panel; top right quadrant), whereas they were not expressed on day
7 NK-1.1- cells (top panel,
bottom right quadrant). The same analysis performed 1
day later (day 8), compared with the day 7 analysis, showed an
increased relative number of cells expressing Ly49A [8 ± 0.6%
versus 5 ± 1.1% (middle panels, top right
quadrants) and 5 ± 0.8% versus 1 ± 0.2%
(middle panels, bottom right quadrants)] or Ly49C/I
[10.1 ± 1.4% versus 5 ± 0.3% (bottom
panels, top right quadrants) and 10.6 ± 3.4%
versus 1.8 ± 0.6% (bottom panels, bottom right
quadrants)]. MFI values show that although more numerous, cells
from day 8 expressed Ly49A and Ly49C/I at low to intermediate levels
(as demonstrated by the decrease of MFI values reported in Table 1
) compared with day 7 cells. This suggests that the day 7
NK-1.1- cells were progenitors which, after 1
more day of culture, started to express both NK-1.1 and Ly49 molecules
not at the high levels of fully mature NK cells but at low to
intermediate levels and, consequently, that expression of Ly49
molecules was a late event in the differentiation pathway of NK cells.
The day 7 analysis in Fig. 4
shows that a minor population of NK cells
was already present (possibly derived from the few
NK-1.1low cells detected at day 3), but the large
expansion of NK cell number was due primarily to the
NK-1.1-CD3- cell
population predominant at day 7 and that this double-negative
population expressed mature NK cell markers starting from day 8. This
further confirms that the generation of NK cells observed here was
sustained by proliferation and subsequent differentiation of
NK-1.1- progenitors and not by mature
NK-1.1+ cells contaminating the original BM
progenitor population.
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shows that a
significantly lower cell number (around 10 times less) was detected in
the group to which the anti-H-2b mAb was added
compared with controls. Cell counts determined that the inhibition of
cell expansion was significant at day 3 (Fig. 7)
. To further control the specificity of the anti-class I mAb inhibition
and to avoid the possibility that the inhibition was due to an
antibody-dependent cell cytotoxicity mechanism, Fc receptors were
blocked by the addition of a blocking anti-Fc receptor to the cultures
at day 0, and this treatment did not revert the anti-class I
mAb-induced inhibition (data not shown). The few cells obtained after
13 days of culture in the group to which the anti-class I mAb was added
were pooled to obtain a sufficient number of cells for flow cytometric
analysis. They were mature NK cells and expressed Ly49A,
Ly49C/I, and Ly49G at the same frequency as controls (moreover, cells
from both experimental and control groups expressed Fc receptors; data
not shown), thus suggesting that the inhibitory effect was only
quantitative and was not directed to a specific
Ly49+ NK cell subpopulation.
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, the mAb exerted the same inhibitory effect in all groups, irrespective
of the haplotype of stromal cells. Taken together, these experiments
suggest that the inhibitory effect of the anti-class I mAb was not
exerted on supportive stromal cells but rather on the
CD44low/-CD2-class
Ilow progenitor population.
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shows that in the group in which the anti-class I mAb was added, the
immature NK-1.1-CD3-
cells were absent, and only the
NK-1.1+CD3- cells were
detected. These experiments clearly suggest that the inhibitory effect
of anti-class I mAb was exerted on the double-negative NK progenitor
and exclude the possibility of an effect exerted on the mature progeny
generated in culture. The correlation between disappearance of the day
7 NK-1.1-CD3- cells and
inhibition of NK cells at day 13 is an additional indirect proof that
day 7 NK-1.1-CD3- cells
are progenitors ready to give rise to the NK cells present at day 13.
It is noteworthy that the relative number of similar day 7
NK-1.1+ cell populations present in Figs. 4
, 6
, and 9
show a certain degree of variability, although the SE analysis
did not find evidence of significant differences among them.
This variability may be explained by the method used to separate the
progenitor cell population from fresh BM. Magnetic bead separation is a
technique that allows a certain degree of variation compared with more
precise techniques such as cell sorter separation. On the other hand,
our method allows the processing of enormous numbers of BM cells in an
acceptable period of time.
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shows that in the control group, two populations were present at day 3:
(a) one expressing little or no class I; and (b)
the other expressing a high level of class I. Interestingly, in
the group in which the anti-class I mAb was added together with IL-2,
only the class Ilow population was detectable.
The FITC-conjugated anti-class I mAb used to analyze cells at day 3 was
different from the mAb added at day 0, and previous trials have
demonstrated that these two mAbs bind to different epitopes and do not
interfere with each other’s binding. These data clearly suggest that
IL-2 and stroma coculture induced generation of class
I+ cells from class Ilow
progenitors and that the anti-class I mAb exerted its effect on these
class I+ progeny. The possibility that class
I+ cells present on day 3 were not derived from
class Ilow cells but were a consequence of
expansion of contaminating class I+ cells that
might not have been completely depleted was investigated by precoating
the original
CD44low/-CD2-class
Ilow cells with the
anti-H-2b mAb before placing them in coculture
with IL-2 at day 0. By this means, any class I+
cell contaminating the original class Ilow
progenitor population would be inhibited by the mAb. Only a very modest
(<10%) inhibition was seen after the precoating of day 0 cells, thus
suggesting that the effect seen when mAb was added to the cultures was
exerted on the class I+ cells derived from class
Ilow early precursor cells after coculture and
stimulation with IL-2 (data not shown).
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The purpose of this work was to examine the role of MHC class I molecules on proliferation and differentiation of NK progenitors able to generate in vitro mature NK cells expressing Ly49 molecules.
To isolate a progenitor population, we took advantage of a previously isolated BM population of NK progenitors characterized as CD44low/-NK-1.1-TCR- that were able to proliferate and differentiate in vitro in the presence of IL-2 on supportive stromal cells (10) . We obtained a CD44low/-CD2- cell population, which is basically a lineage-negative population because most mature hematopoietic cells are CD44high, and T, B, and NK cells also express CD2 on their surfaces. It has been shown previously (17) that hematopoietic stem cells express high levels of class I molecules but that class I molecules expression is down-regulated as they differentiate to more committed progenitors. To see whether this was also the case for NK precursors, CD44low/-CD2-class Ilow cells were tested for the presence of NK progenitors. Furthermore, because all mature BM cells, including NK cells, express class I molecules on their surface, depletion by anti-class I mAb represents an additional step for eliminating mature NK cells or other lineage-positive cells still present in the isolated population. Thus, contamination of mature NK cells in the CD44low/-CD2-class Ilow population that would have given false positive results was avoided by the combination of three mAbs directed against CD44, CD2, and class I and highly expressed on mature NK cells. The use of these mAbs in three sequential steps eliminated any NK cells remaining after the previous depletion step. The result of this procedure was tested in two ways using a phenotypic assay and a functional assay. Flow cytometric analyses showed that cells were NK-1.1-, DX5-, and Ly49G2-. Furthermore IL-2-induced expansion of mature NK cells undetectable by flow cytometry did not occur because our population did not grow after stimulation with IL-2 alone but only after stimulation with IL-2 and stroma, and the stroma dependence is a feature of progenitors but not of mature NK cells (9) . Thus, unlike mature NK cells, our precursor population was stroma dependent.
The tested progenitor population gave rise to mature NK cells expressing Ly49A, Ly49C/I, and Ly49G. In a recent work by Williams et al. (9) , Ly49s+ NK cells were obtained after two subsequent steps of culture. During the first step, their progenitor population gave rise to Ly-49- cells after stimulation with different cytokines such as IL-7, SCF, and flt3L, but without stroma. The obtained Ly49- cells completed their differentiation by expressing Ly49s molecules when stimulated with either IL-2 or IL-15 (used indifferently) and stroma. Compared with this work, our progenitors seem to be in a later step of differentiation because they required IL-2 and stroma for differentiation [which were used by Williams et al. (9) in the second step of differentiation], but not IL-7 and other "first-step" growth factors. Of interest, NK cells generated in our culture system display a similar proportion of Ly49s as fresh splenic NK cells, in contrast with the Ly49s proportion of NK cells generated in the study of Williams et al. (9) . In our opinion, the mentioned contrast is only apparent and may be due to the different types of stroma used. We used syngeneic stroma (both progenitors and stroma derived from mice with a H-2b haplotype) that would represent a more physiological model, whereas Williams et al. (9) used an allogeneic stroma (H-2K from OP mice cocultured with H-2b progenitors) that may alter the Ly49 repertoire as hypothesized in the "Discussion" section of the same work (9) . Moreover, the possibility that BM stromal cells are able to influence the NK cell repertoire is also hypothesized in a study by Sykes et al. (5) and was recently demonstrated by Roth et al. (18) .
The time course flow cytometric analysis (day 3, 7, and 13) showed that after 7 days, about 10–20% of cells were mature NK-1.1+CD3- cells, whereas the large majority (80–90%) of cells did not express NK-1.1, CD3, Ly49A, Ly49C/I, or other differentiation markers. This suggested that the major population was derived from progenitors that underwent an extensive proliferation, but not differentiation. The undifferentiated day 7 population expressed Ly49A, Ly49C/I, and low levels of NK-1.1 after 1 additional day of culture (on day 8), demonstrating their ability to differentiate to mature NK cells. Expression of Ly49A and Ly49C/I on day 8 suggests that these molecules are expressed as a late event in the differentiation pathway and expressed simultaneously with or immediately before NK-1.1 molecules. The presence of at least two cell types with different characteristics has also been shown previously (19) in humans in a lineage-negative progenitor population that seems to be similar to the one isolated by us in the mouse.
Once we standardized the experimental model, the second set of experiments was done to examine the influence of MHC class I antigens on in vitro differentiation of CD44low/-CD2-class Ilow cells. The anti-H-2b mAb was strongly inhibitory and did not exert its effect by acting on stromal cells because inhibition was seen even when the generation of NK cells was supported by allogeneic stromal cells derived from C3H mice (H-2k) or by H-2b-deficient stromal cells from Tap-1-/- mice that could not have been recognized by the anti-H-2b mAb. Taken together, these data suggest that the anti-H-2b mAb exerted its effect on the progenitor population, and flow cytometric analysis on day 7 cells clearly indicated that only its undifferentiated NK-1.1-CD3- but not the differentiated NK-1.1+CD3- progeny was absent after the addition of mAb to cultures. The data obtained with the mAb preadhered to precursors suggest the presence in the original CD44low/-CD2- cells of a class Ilow population that can differentiate within 3 days to a class I+ subpopulation. We do not know what the mechanism of the inhibitory effect of anti-class I mAb was. It is not probable that it blocked the binding to cells expressing class I ligands on their surface because the only known ligands for class I molecules are expressed on mature NK cells (Ly49 molecules) or on T cells (CD8 molecules), and no such cells were present in the first 3 days of culture after the addition of IL-2, when the anti-H-2b mAb had already exerted its inhibitory effect. Instead, we favor the hypothesis of a stimulatory effect of the mAb on class I molecules. This hypothesis is based on previous data showing that class I molecules are not only ligands that trigger T lymphocytes through the TCR or inhibit/stimulate NK cells trough Ly49 molecules but are also able to mediate different functions in the cell on whose surface they are expressed. For example, MHC class I molecules, when stimulated, may transduce signals (20 , 21) , regulate cell adhesion and proliferation (22) , and induce apoptosis (23) . This potential opens the possibility for class I molecules to interfere with cell functions and, as a consequence, with physiological processes not sufficiently analyzed up to now. For example, stimulation of class I molecules may determine the inhibition demonstrated in this work by inducing either apoptosis, growth arrest, or both of NK cell progenitors, thus controlling the NK cell number in the BM and in the periphery. Moreover, because it has also been shown that class I is linked with the receptor of IL-2 (24) , it may be possible that these molecules, when stimulated by the anti-H-2b mAb, interfered with signals triggered by stimulation of the IL-2 receptor and, as a consequence, inhibited the triggered IL-2-dependent proliferation program. The aforementioned hypothesis translated in a more general context would also suggest that the development of NK cell progenitors to mature NK cells is under the control of mature BM cells expressing ligands for H-2 (CD8 or Ly49s on the surface of T lymphocytes or NK cells, respectively) that mimic the inhibitory function of class I mAb. An additional support for this possibility is our previous data showing that BM T cells are able to inhibit NK cell differentiation (25) . Although appealing, this hypothesis would require additional testing, which is currently in progress.
Although the normal number of NK cells in class I-deficient mice such as Tap-1-/- or ß2-microglobulin-/- mice may appear to be in contrast with the inhibitory role of class I molecules shown in the present study, this is only an apparent discrepancy. Genetic absence of class I molecules cannot necessarily be mimicked by binding of a specific anti-class I mAb. In class I-deficient mice, the absence of class I molecules may trigger compensatory mechanisms, and the consequences of the lack of their expression may affect NK cell differentiation by acting from the beginning of the hematopoietic process (e.g., on totipotent hematopoietic stem cell) until the ultimate events; on the other hand, the anti-class I mAb effect shown by us is exerted specifically on IL-2-responsive, stroma-dependent progenitors ready to differentiate to mature NK cells within 1–2 weeks. In addition to this, the mAb may be either stimulating or blocking (as discussed above) class I molecules, and this cannot be seen in class I-deficient mice because the protein is not expressed. Finally, the role of control in NK cell differentiation hypothesized by us would explain why changes in expression of MHC class I antigens occur on hematopoietic progenitors during their differentiation as shown in the present study or in previous reports (17) . Because of this, we think that the in vitro model used by us is complementary to the knockout mice models and analyzes a different function of class I molecules.
Our studies suggest that modulation of class I molecule expression on surface of progenitors may represent a mechanism by which progenitors can control their number before completing the differentiation program. Furthermore, these data suggest the possibility that class I molecules expressed on progenitors play a role in NK cell differentiation. Likewise, class I molecules expressed on surrounding cells play a role in cellular adaptation mechanisms.
In conclusion, our data, in addition to the work on class I-deficient mice, highlight class I molecules as keys to the control of NK cell development by different mechanisms and/or in different phases of their developmental process. We think that our experimental model is a useful tool to consider a possible mechanism to modulate the number of NK cells by acting on class I molecules of specific progenitors.
Materials and Methods
Animals.
C57BL/6 and C3H/HEN mice were purchased from Charles River (Calco,
Lecco, Italy). Tap-1-/-
mice were purchased from The Jackson Laboratory
(Bar Harbor, ME). Mice were used at 2–4 months of age. They were
housed in an isolated colony and fed laboratory chow and acidified (pH
2.4) water ad libitum.
Culture Medium.
The standard culture medium was RPMI 1640 (Life Technologies, Inc.,
Grand Island, NY) supplemented with 2 mM
L-glutamine, 100 units/ml penicillin, and 100 mg/ml
streptomycin. For NK-long term BM cultures, 5% FCS (Life Technologies,
Inc.) and 5 x 10-5 M
2-Mercaptoethanol were added to the standard culture medium
(complete medium).
BM Cell Harvest.
BM was obtained from the femurs and tibias of mice killed by cervical
dislocation that were disinfected by immersion in 70% ethanol. After
the bones were removed and cleaned of skin and muscle, the BM was
exposed by cutting the ends of the bones and expelled by inserting a
needle and forcing medium through the bone shaft. The cell clumps were
then broken by vortexing, and an aliquot of the cell suspension was
counted with a hemocytometer.
Cultures.
BM cells were diluted in complete medium at a concentration of 2.5 x 106 cells/ml. Aliquots of 2.5 x
105 cells/well in 0.1 ml of complete medium were
plated in each of the 60 innermost wells of round-bottomed 96-well
plates (Costar; Corning Inc., Corning, NY), and the remaining external
wells were filled with sterile water. Cultures were incubated at 37°C
in 5% CO2 in humidified air without further
medium change for 6 weeks. To obtain pure supportive stromal cells,
after 6 weeks, the cultures were exposed to 20 Gy to kill the
hematopoietic cells (including NK precursors) while sparing a sparse
layer of viable and functional stromal cells. The cells containing the
NK cell precursors (see below) were seeded at a concentration of 1 or
5 x 103
onto the stroma, and IL-2 (500
IU/ml) was added (day 0). The generated cells were then harvested and
tested after various days (day 3, day 7, day 8, and day 13).
Isolation of the NK Cell Precursor-containing Population.
BM cells from six mice were depleted of RBCs by incubation for 5 min at
room temperature with 4 ml of autoclaved RBC lysing buffer (8.32 grams
of NH4Cl, 0.84 gram of
NaHCO3, and 0.043 gram of EDTA per liter of
deionized distilled water). The cells were then pelleted, and the
FITC-conjugated anti-CD44 mAb (0.006 µg/million BM cells) was added.
The cells were incubated at 4°C for 20 min in the dark, washed twice,
and resuspended in 1 ml of 1x PBS with 10% FCS. The cells were then
added to anti-FITC-conjugated magnetic beads (PerSeptive Diagnostic,
Cambridge, MA) contained in 5 ml tubes after the suspension
medium was washed out and incubated for 30 min in a rotor at 4°C.
After incubation, the tube of cells was exposed to a Dynal MPC-1 magnet
(Dynal, Great Neck, NY), and the cells remaining in suspension were
collected and incubated for a second step with beads. The anti-CD44 mAb
was used at a very low concentration (0.006 µg/million target cells)
to deplete the CD44high but not the
CD44low BM cells. Alternatively,
CD44high BM cells can be eliminated by a step of
magnetic bead depletion using the anti-Gr-1 mAb (1 µg/ml target
cells), which is expressed on myeloid cells that also express CD44 at
high intensity. The CD44low/- BM cells were then
depleted of CD2+ by a similar step of depletion
using a high concentration of FITC-conjugated anti-CD2 mAb (1 µg/ml
target cells) to achieve the complete elimination of
CD2+ BM cells. The third step was modified
slightly: the FITC-conjugated anti-class I mAb (1 µg/ml target BM
cells) was preincubated for 20 min at 4°C with 1 ml of washed
magnetic beads. At the end of the incubation period, the uncoated mAb
was washed out, and the
CD44lowCD2- cell
suspension was added to the anti-class I mAb-coated magnetic beads and
incubated as described for the previous steps to eliminate only class
Ihigh cells.
Cell Count.
The cells were counted with a hemocytometer, and live cells were
identified by using the trypan blue exclusion assay.
Cytokines.
Human recombinant IL-2 was generously provided by Hoffmann La Roche
(Nutley, NJ) and used at a concentration of 500 IU/ml.
Antibodies.
The following mAbs were used: (a) FITC-conjugated rat
antimouse CD44 (IgG2b; clone IM7); (b) FITC-conjugated rat
antimouse CD2 (IgG2b; clone RM2-5); (c) FITC-conjugated
mouse antimouse H-2Kb (IgG2a; clone AF6-88.5);
(d) purified mouse antimouse
H-2Kb/H-2Db (IgG2a; clone
28-8-6); (e) purified mouse antimouse isotypic control
(IgG2a, 
; clone G155-178); (f) purified mouse antimouse
CD45.2 (IgG2a; clone 104); (g) FITC-conjugated hamster
antimouse CD3
(CD3; IgG; clone 145-2C11); (h)
FITC-conjugated hamster isotypic control (IgG; clone UC8-4B3);
(i) PE-conjugated mouse antimouse NK-1.1 (IgG2a; clone
PK136); (j) PE-conjugated mouse isotypic control (IgG2a;
clone G155-178); (k) PE-conjugated rat antimouse Pan-NK
cells (IgM; clone DX5); (l) FITC-conjugated rat antimouse
Ly49G2 (LGL-1; IgG2a; clone 4D11); (m) FITC-conjugated mouse
antimouse Ly49A (IgG2a; clone A1); and (n) FITC-conjugated
mouse antimouse Ly49C/I (IgG2a; clone 5E6). All mAbs were purchased
from PharMingen (San Diego, CA).
Flow Cytometry.
Approximately 0.5–1 x 105 cells (<5% of
dead cells/sample) were pelleted in a round-bottomed centrifuge tube at
200 x g for 5 min. The pellet was resuspended in 10
µl of the predetermined dilution of the mAb and incubated in the dark
at 4°C for 20 min. The cells were washed twice and resuspended in 1x
PBS. The samples were then analyzed using a FACScan flow cytometer
(Becton Dickinson, Sunnyvale, CA). Dead cells were gated out by size
(forward scatter). The percentage of positive cells was
calculated after subtraction of the background present in the isotypic
control sample.
Statistics.
Data are presented as mean ± 1 SE. Student’s t test
was used for all experiments. Values of P > 0.05 were
considered to be not significantly different (significance was defined
as P < 0.05).
Acknowledgments
We thank Sallie S. Boggs and Kenneth D. Patrene for helpful criticism and for reviewing the manuscript. This study is dedicated to the memory of Rosalba Moraca.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by the Ministero
dell’Università e Ricerca Scientifica e Tecnologica, the
Associazione Italiana Ricerca sul Cancro, and Progetlo
Finalizzato Biotecnologie. 
2 To whom requests for reprints should be
addressed, at Department of Clinical and Experimental Medicine, Section
of Pharmacology, University of Perugia, Via del Giochetto, 06100
Perugia, Italy. Phone: 39-75-5857493; Fax: 39-75-5857405; E-mail: farmaco{at}unipg.it
3 The abbreviations used are: NK, natural killer;
BM, bone marrow; IL, interleukin; mAb, monoclonal antibody; TCR, T-cell
receptor; MFI, mean fluorescence intensity.
Received for publication 8/15/00. Revision received 11/15/00. Accepted for publication 11/16/00.
References
2 domain in regulation of cell adhesion and proliferation. J. Immunol., 156: 1415-1424, 1996.[Abstract]
-chain, and induces apoptosis. J. Immunol., 158: 3189-3196, 1997.[Abstract]
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,
CD44low/-CD2-class Ilow BM cells
stimulated by IL-2 and cultured without supportive stromal cells. The
results from four independent experiments are expressed as the
mean ± 1 SE.
), the
anti-H-2b mAb (
), or an anti-CD45.2 mAb (
),
respectively, is shown. Results are the number of
cells/well ± 1 SE of seven independent experiments.
