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H-2b and H-2b H-2d Bone Marrow Chimeras1
Department of Clinical Immunology, Medical School Hannover, Hannover, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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H-2b chimeras Ly49A+,
Ly49C/I+, and Ly49A+/Ly49C/I+
proportions were mainly diminished upon interaction with cognate
ligands. Also in H-2b H-2d chimeras,
Ly49A+ and Ly49A+/Ly49C/I+ subsets
were reduced, but there was a transient normalization of
Ly49C/I+ proportions in the noncognate host. After 120 days
all subsets were reduced. Therefore, down-regulation of developing
Ly49A+ and Ly49C/I+ chimeric NK cell
frequencies by cognate ligands within 7–8 wk after BM transplantation
may be important for successful engraftment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At least nine genes of the mouse Ly49 multigene family have been identified so far (19). The Ly49 molecules are thought to play a decisive role in determining the specificity of NK cells through recognition of class I molecules and subsequent delivery of inhibitory and in two cases, Ly49D and Ly49H (20, 21), activitory signals. The cognate Ags for Ly49A are H-2Dd and Dk. Ly49C/I recognizes the Ags Kb, Kd, and Dd. Ly49C alone of C57BL/6 (B6) mice recognizes H-2Kb whereas Ly49C of BALB/c mice recognizes H-2Kb but also H-2Kd and H-2Dd (22, 23). The ligand for Ly49I is still unknown. Ly49I is not expressed by BALB/c mice. The Ly49 molecules define separate NK cell subsets but are partially coexpressed and form overlapping subsets (24); other NK cells do not express known Ly49 at all. The important question of how NK cells acquire specificity and tolerance during maturation and within a naturally MHC-varying environment is still unanswered. Evidence for an influence by host MHC class I has been put forward (14, 25, 26, 27, 28). It seems inconsistent that, for example, very similar frequencies of cells expressing a certain receptor, even a potentially harmful one, are observed in completely MHC class I-different mice (14). It is postulated that after an initial random expression of Ly49 receptors (29) and generation of a diverse repertoire (30), cells are selected via MHC expression levels of the host (31). Potentially harmful cells are either deleted by clonal selection or adapt by becoming anergic or having an altered specificity (13). It was shown that Ly49A+ NK cells from H-2b mice showed reduced killing of H-2b Con A blasts compared with those from H-2d mice but showed efficient killing of ß2m- lymphoblasts (13).
The production of fully allogeneic MHC-disparate, for example
H-2d and H-2b, chimeric
mice poses a challenge to donor and recipient NK cells developing in
mixed MHC class I hematopoietic hosts. Changes of expression levels in
this context rather than expression frequencies have been described so
far (32, 33). Recently the numbers of
Ly49A+, Ly49C+, and
Ly49G2+ NK cell subsets were also addressed in
[BALB/c B6] chimeras (34) at 6 wk after BM
transplantation (BMT).
In this study, we examined the in vivo development of
Ly49+ subset frequencies of peripheral NK cells
at 5-day intervals to determine alterations over time within the
critical phase of engraftment. We also analyzed whether these newly
acquired patterns remained stable in the surviving mice. We focused on
Ly49A and Ly49C/I receptors, analyzing donor and host NK cells
separately in [BALB/c B6] as well as [B6 BALB/c] chimeric
mice. The Ly49 receptor repertoire alterations found are discussed in
the context of the external/internal MHC class I-dependent calibration
model recently proposed by Kåse et al. (15). Hence, the
present study is an approach toward understanding NK cell tolerance in
an allogeneic BMT model and thus might provide a basis for further
improvement of the engraftment process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal inbred BALB/c (H-2d) and C57BL/6 (H-2b) mouse strains were used as an in vivo system for allogeneic and syngeneic BMT. BALB/c and B6 mice were bred in the animal facilities of Hannover Medical School (Hannover, Germany), maintained under germ-free conditions, and used at the age of 8–12 wk. An oral antibiotic prophylaxis with sulfamethoxazole and trimethoprim in the drinking water of donor and recipient mice was started 3–4 days before irradiation and continued for 60 days. Animals with four indicators of graft-vs-host disease (GvHD) (alopecia, scleroderma, weight loss, hunched posture, severe diarrhea, bleeding, or inflammation of the eyes or nose) were suspected of presenting GvHD and underwent histological examination.
Abs and reagents
Currently available Ly49 mAbs (35) such as FITC- or
PE-conjugated anti-Ly49A-FITC (A1), anti-Ly49C/I-PE (5E6) as
well as anti-NK1.1-FITC and -PE (PK136), anti-DX5-FITC and -PE
(DX5, a pan-NK cell marker), anti-H-2Kb
(AF6–88.5), and anti-H-2Kd (SF1–1.1) were
purchased from PharMingen (Hamburg, Germany). Ly49C/I mAb recognizes
both Ly49C and Ly49I (36). A1 does not label the BALB/c
allele of Ly49A but labels the B6 allele (21). B6 NK cells
were distinguished from BALB/c cells by using
anti-H-2Kb as well as anti-NK1.1 reacting
with B6 but not BALB/c NK cells. Isotype-matched mouse IgG2a mAbs were
used as negative staining controls. To block Fc
II/III
receptor-mediated unspecific binding, anti-FcII/III (2.4G2) from
purified hybridoma supernatants was used. The Ab-producing hybridoma
was obtained from American Type Cell Collection (Manassas, VA). To
eliminate T lymphocytes in the graft, anti-Thy-1.2 was applied as
hybridoma supernatant (the hybridoma was kindly provided by S. Izui,
Geneva, Switzerland) as well as low toxicity rabbit complement,
purchased from Cedarlane Laboratories (Hornby, Ontario, Canada).
BM transplantation
BM cells were flushed out from femur and tibia of two donor
animals per five recipients under aseptic conditions. Stem cells were
enriched via a Ficoll-gradient, washed in Dulbecco’s PBS, and depleted
of T cells by incubation with anti-Thy-1.2 followed by low toxicity
rabbit complement according to the manufacturer Cederlane Laboratories.
Cell suspensions were adjusted with Dulbecco’s PBS to 
5–8 x
106 BM cells/inoculum and kept on ice. The
recipient B6 mice were exposed to a lethal dose of 1200 rad of
gamma-irradiation from a Cs137 source, given in
two doses of 600 rad each, 5–6 h apart. The recipient BALB/c mice were
exposed to a lethal dose of 950 rad in two doses of 475 rad. Within the
next 2 h, they were inoculated with syngeneic and allogeneic BM
cells of sex- and age-matched B6 and BALB/c mice, respectively
(designated [donor recipient]). After i.p. narcotization, the BM
cell suspensions were injected into the tail veins. The relatively
small number of 5–8 x 106 graft cells was
chosen in order not to override the recipient’s reaction
(37) and to achieve a stable 80–90/10–20%
donor/recipient chimerism.
B6 mice were transplanted with allogeneic BALB/c BM (n
= 15; [BALB/c B6]) or syngeneic B6 BM (n = 10;
[B6 B6]). BALB/c mice were transplanted with allogeneic B6 BM
(n = 13; [B6 BALB/c]) or syngeneic BALB/c BM
(n = 10; [BALB/c BALB/c]). The syngeneic BMTs
served as controls. All experiments were performed twice with five
(eight in one case) recipients each. The [BALB/c B6] BMT was
performed three times.
After syngeneic [B6 B6] BMT, 8 of 10 mice (80%) survived
long-term (>120 days); one death after 40 days was unrelated to BMT.
Five of 10 mice (50%) survived long-term after syngeneic [BALB/c
BALB/c] BMT. After allogeneic [BALB/c B6] BMT, 4 of 15 mice
(27%) survived >120 days; 33% had been alive until day 40. Six of 13
(46%) BALB/c mice survived long-term after allogeneic [B6
BALB/c] BMT; 9 were alive until day 44 (69%). Most of the animals
succumbing 2 wk after allogeneic BMT showed alopecia, weight loss,
hunched posture, diarrhea, and inflammation of the eyes as indicators
of GvHD, but other causes like rejection, engraftment failure,
infection, and rivalry were also suspected due to signs of anemia,
bleeding, and skin injuries. Histology rarely showed GvHD. Only data of
surviving animals were included.
Preparation of peripheral NK cells and flow cytometry (FCM)
A total of 100–200 µl of blood was taken from the retroorbital plexus every 5 days for 8 wk after BMT, starting on day 10. Approximately 120 days after BMT, the experiment was finished with a final single measurement.
Blood was taken from 15 BALB/c and B6 mice, each for investigation of pretransplant Ly49 expression patterns of peripheral NK cells. Nucleated cells were enriched by hypotonic lysis of erythrocytes and washing with PBS/1% BSA. After preincubation with 2.4G2 mAb and mouse serum at a dilution of 1:10 for 10 min, they were incubated for 0.5 h with the respective primary Abs at a dilution of 1:40, fixed in 1% formaldehyde, and then analyzed on a flow cytometer (Becton Dickinson FACScan, San Jose, CA). Negative controls with PBS/1% BSA instead of a primary Ab were performed for each mouse. Forward and side scatter were used to gate on the lymphocyte population. Between 5.000 and 15,000 gated events were collected for analysis. Data analysis was performed by using CellQuest (Becton Dickinson FACScan) or Windows Multiple Document Interface for Flow Cytometry (WinMDI, Scripps Research Institute, La Jolla, CA) software. The two-tailed Student’s t test for comparison of means with unequal variances was applied (Microsoft Excel software, Redmond, WA). Differences were considered significant if p < 0.05 and highly significant if p < 0.01.
Calculation of pretransplant Ly49 expression frequencies
Pretransplant baseline expression frequencies were calculated in
two ways. For comparison of mean
DX5+/Ly49+ subsets,
pretransplant individual B6 and BALB/c NK cell frequencies were
calculated together (see Figs. 2
, 3, 5, and 6). For separate analysis
of recipient or donor NK cells, means of B6 and BALB/c
DX5+/Ly49+ NK cell
frequencies were considered (see Fig. 4). Ly49AB6
values were referred to because mAb A1 did not detect
Ly49ABALB/c.
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To test chimerism of NK cells after allogeneic BMT, two-color fluorescence staining of the total NK cell population with anti-DX5 and anti-H-2Kb or anti-H-2Kd was performed on each day of measurement. Thus engraftment success or failure was followed continuously. Syngeneically transplanted mice were used as controls.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice solely express the MHC class I haplotype
H-2d whereas B6 mice express
H-2b. Recipient or donor NK cells expressing the
respective MHC class I molecule were detected by double
immunofluorescence staining of the Ags H-2Kd or
H-2Kb and DX5. In lethally irradiated mice of
both strains, recipient NK cells were found in an initially decreasing
(50–60% after 10 days, 30–40% after 20–30 days, and 20% after 30
days; data not shown), but then relatively stable proportion of
10–20% of all NK cells (Fig. 1). Donor
NK cell numbers were increasing to 50% of the total NK cell
population within the first 10 days, reaching 70% after 20–30
days, 80% after 30–40 days (data not shown), and 80–90% after 55
days (Fig. 1). This chimeric pattern remained stable up to 120 days,
varying <10%. Hence, the Ly49 expression patterns of both recipient
and donor NK cells during engraftment could be analyzed separately in
mixed allogeneic BM chimeras over a long posttransplant period.
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Modulation of donor Ly49A+/H-2b peripheral NK cell frequency in a new but cognate H-2d host
Ly49ABALB/c and
Ly49AB6 both recognize
H-2Dd and H-2Dk ligands,
but not H-2Db ligands. Therefore, there is no
known ligand for Ly49A in B6 H-2b mice. To
investigate the influence of a new but cognate host environment on the
expression frequency of Ly49A on H-2b donor NK
cells, we transplanted B6 BM into BALB/c mice (Fig. 2
, A and C). The
frequencies of the Ly49A-expressing NK cell subsets after allogeneic
BMT are shown together with the syngeneic B6 controls ([B6 B6]
BMT) starting from the pretransplant baseline values and ending 55 days
after transplantation. The allogeneic frequencies were compared with
the syngeneic on day 55. Because mAb A1 only recognized the Ly49A
allele of B6 but not BALB/c mice, the
Ly49A+/DX5+ NK cell
population was congruent with the B6 donor-derived NK cell population.
B6 specifically labeled NK1.1+ NK cells were
investigated separately to check whether there was a difference
compared with the results obtained with anti-DX5 (Fig. 2, C and
D).
Before BMT, Ly49A was expressed on 16.5 ± 4.1% of DX5+ B6 NK cells and on 19.8 ± 3.6% of NK1.1+ B6 NK cells (p > 0.05, not significant; <1% of the gated B6 lymphocyte population was NK1.1- DX5+ in naive and syngeneically transplanted B6 mice).
As shown in Fig. 2, A and C, only the
allogeneic BMT clearly resulted in altered expression frequencies of
the donor Ly49A+ NK cell populations compared
with the B6 syngeneic controls. After allogeneic BMT, the proportion of
Ly49A-expressing NK cells declined already after 15–20 days
post-BMT to a significantly lower level of 4.9 ± 1.7%
Ly49A+/DX5+ NK cells (Fig. 2A) and 5.4 ± 1.7%
Ly49A+/NK1.1+ NK cells on
day 55 (Fig. 2C) compared with day 0 (19.8 ± 3.6%)
and compared with the B6 syngeneic controls, which continued expressing
the pretransplant expression pattern (day 55: 12.6 ± 1.5%
Ly49A+/DX5+ NK cells;
17.2 ± 2.9%
Ly49A+/NK1.1+ NK cells).
Thus interaction with a new but cognate H-2d
ligand of the host environment down-regulates the donor
Ly49A+ H-2b NK cell
frequency in peripheral blood.
Modulation of recipient Ly49A+/H-2b NK cell frequency by a new but cognate H-2d donor ligand
It was then studied whether the exposure of recipient NK cells to
a new but cognate ligand just on donor BM-derived cells, and not the
whole microenvironment of the host, was sufficient to lead to a
reduction of peripheral Ly49A + NK cells (Fig. 2, B and D). We performed a transplantation of
BALB/c H-2d BM to a B6 H-2b
background and examined the frequency of Ly49A expressing
recipient-derived NK cells. Again, we found a lower frequency of
Ly49A+ NK cells only after allogeneic [BALB/c
B6] BMT but not syngeneic BMT. The proportion of
Ly49A+/DX5+ NK cells
constantly decreased from pretransplant levels and was significantly
reduced after 55 days (1.5 ± 0.7%, Fig. 2B) compared
with the B6 syngeneic controls (11.0 ± 4.8%, Fig. 2B). The syngeneic controls kept on expressing the
pretransplant expression pattern without significant changes (day 0 vs
day 55: p = 0.08,
Ly49A+/DX5+;
p = 0.16,
Ly49A+/NK1.1+). Using the
NK1.1 B6 NK cell-specific marker, a significant proportional decrease
of the recipient
Ly49A+/NK1.1+ NK cell
numbers was also observed compared with the syngeneic controls (Fig. 2D). Interestingly, this reduction of the recipient-derived
Ly49A+/NK1.1+ subset in
[BALB/c B6] BMT occurred later, after 45 days, compared
with the donor-derived
Ly49A+/NK1.1+ and
Ly49A+/DX5+ NK cell subset
in [B6 BALB/c] BMT, which had declined early after 20 days. The
reduction of the recipient DX5+ population after
[BALB/c B6] BMT was less delayed after 25 days in contrast to the
NK1.1+ population. Therefore donor and recipient
NK cells developed different Ly49 subset frequencies at different
times.
In summary, interaction with a new but cognate
H-2d donor ligand on BM-derived cells also
down-regulated Ly49A+ recipient
H-2b NK cell frequency effectively in a known
host environment. After 30–40 days, the immune reconstitution of
the donor-derived hematopoietic cells had taken place for the most part
and 80% donor-derived cells were found in the peripheral blood
(Fig. 1). The delayed reduction of the recipient NK cells indicated
that the amount of ligand-expressing cells was relevant for this
decrease.
Modulation of donor Ly49C/I+/H-2d NK cell frequency in a new but cognate H-2b host
Parallel to Ly49A, we examined the expression frequency of
Ly49C/I+ NK cells. B6 NK cells express Ly49C/I
whereas BALB/c NK cells only express Ly49C.
Ly49CBALB/c recognizes the
H-2Kb, H-2Kd, and
H-2Dd ligands (36, 38).
Ly49CB6 only recognizes
H-2Kb. It was investigated whether the
Ly49C/I+ NK cell frequency was also
down-regulated upon interaction with a cognate ligand as was seen for
Ly49A. The transplantation of BALB/c BM to the B6 background [BALB/c
B6] led to an interaction of the donor H-2d
NK cells with a new but cognate ligand, H-2Kb,
expressed in the host environment (Fig. 3, A and C).
Considering our results on Ly49A-expression frequencies, a
down-regulation of Ly49C-expressing BALB/c NK cell proportions could be
expected.
Before BMT Ly49C/I was expressed on 37.9 ± 7.7% of
DX5+ NK cells, means of both strains were
calculated together. On either recipient or donor,
NK1.1+ B6-derived NK cells alone Ly49C/I was
expressed on 38.4 ± 6.9% NK cells. 35.8 ± 10.2% of BALB/c
DX5+ NK cells expressed Ly49C (Fig. 4), revealing no statistical difference
between BALB/c and B6 mice.
As shown in Fig. 3A, the proportion of total
donor/recipient
Ly49C/I+/DX5+ NK cells
rapidly decreased after both allogeneic [BALB/c B6] BMT and
syngeneic [BALB/c BALB/c] BMT within the first 3 wk. In the
syngeneic controls (also of [B6 B6] BMT), they subsequently
regained pretransplant frequencies (25 ± 9% on day 55) whereas
after allogeneic BMT they remained significantly reduced after 55 days
(8.2 ± 0.7%). The syngeneic controls exhibited no significant
difference of Ly49C/I+ subsets between day 0 and
day 55 (p > 0.05 B6 and BALB/c). The reduction
of the total donor/recipient
Ly49C/I+/DX5+ NK cell
subset implied that the donor proportion within this population must
have been greatly reduced as they represent 80% of peripheral NK
cells at this time. Additional separate analysis of the donor-derived
Ly49C+/NK1.1-/DX5+
subset on day 50 revealed a more pronounced, statistically significant
proportional reduction of the donor than of the recipient NK cells
compared with day 0 (Fig. 4).
Therefore, parallel to the reduction of the Ly49A+ subset a reduction of the donor Ly49CBALB/c/H-2d NK cell frequency in a new but cognate H-2Kb-expressing host environment was also observed.
Modulation of donor Ly49C/I+/H-2b NK cell frequency in a new and noncognate H-2d host
In contrast to donor Ly49CBALB/c NK cells in
a H-2b host, donor
Ly49C/IB6/H-2b NK cells
developing in the BALB/c H-2d host encounter a
noncognate ligand in the host environment because
Ly49C/IB6 NK cells only recognize
H-2Kb. As expected the proportion of total
donor/recipient Ly49C/I+ NK cells decreased
within the first 2–3 wk but then developed toward pretransplant
numbers (37.9 ± 7.7%,
DX5+/Ly49C/I+) 55 days
after syngeneic (26 ± 7%) as well as after allogeneic (31.2
± 3.3%) BMT (Fig. 3B). Analyzing the
Ly49C+/NK1.1+ NK cells
separately confirmed the normalization for the donor subset directly,
which exhibited a development of subset frequency very similar to the
syngeneic controls (Fig. 3D). Regarding the time points of
variation, the early increase of the total donor/recipient
Ly49C/I+/DX5+ proportion
after 20–25 days to nearly pretransplant levels (Fig. 3B)
was observed similarly in the donor-derived
Ly49C/I+/NK1.1+ NK cell
subset (28.9 ± 2.6%) (Fig. 3D).
This normalization of Ly49C/IB6 donor NK cell frequency in a noncognate host environment confirmed the opposite down-regulating effect of new but cognate ligands.
Modulation of recipient Ly49C/I+/H-2b NK cell frequency in a H-2b host upon interaction with a new and noncognate H-2d donor ligand
Confronting the recipient Ly49C/IB6 NK cells
with the new H-2d donor meant interaction with a
noncognate ligand because they only recognize
H-2Kb. Therefore we would not expect a
down-regulation. However, the recipient
Ly49C/I+/NK1.1+ NK cells
were also markedly reduced after 25 day up to 55 days. Again, the
recipient-derived
Ly49C/I+/NK1.1+ NK cells
had decreased later (Fig. 3C) compared with the constant
decrease 10 days earlier of the total donor/recipient
Ly49C/I+/DX5+ NK cell
population (Fig. 3A). Furthermore, the
Ly49C/I+/NK1.1+ subset
decreased earlier compared with the recipient
Ly49A+/NK1.1+ subset, which
was reduced after 45 days.
Thus, in contrast to the reductions upon encounter of new and cognate ligands, we observed a down-regulation of recipient Ly49C/IB6 NK cell frequency here facing a new but noncognate H-2d donor ligand in a cognate H-2b microenvironment.
Modulation of recipient Ly49C/I+/H-2d NK cell frequency in a H-2d host upon interaction with a new but cognate H-2b donor ligand
In contrast to recipient Ly49C/IB6 NK
cells, recipient
Ly49CBALB/c/H-2d NK cells
were expected to recognize the new H-2Kb donor
ligand after [B6 BALB/c] BMT. As indirectly shown in Fig. 3B, the early reductions of the total donor/recipient
Ly49C/I+/DX5+
proportions implied a reduction of the recipient subset
constituting 40–50% of peripheral NK cells 15 days after BMT.
The separate analysis of the recipient
Ly49C/I+/NK1.1-/DX5+
subset on day 50 showed that they remained on a significantly lower
level (10.7 ± 6.9%, Fig. 4) compared with the donor-derived
Ly49C/I+ subset (27.5 ± 3.2%, Fig. 4) that
was also reduced but closer to pretransplant levels (38.4 ±
7.2%, Fig. 4). This difference was also implicit in the total
donor/recipient
Ly49C/I+/DX5+ NK cell
population that showed a relatively high variation on day 50 (Fig. 3B). Thus donor- and recipient-derived NK cells revealed
statistically significant different Ly49C/I+
subsets on day 50 post-BMT (Fig. 4).
Therefore, the recipient Ly49CBALB/c/H-2d NK cells were down-regulated upon interaction with the new but cognate H-2Kb donor ligand on B6 BM-derived cells.
Long-term maintenance of expression patterns after BMT
A final blood sample was taken from all mice after 120 days to
compare the expression patterns with those after 55 days. As seen in
the second and third columns of Figs. 5
and 6, most significant variations found
55 days after allogeneic BMT still occurred in all mice after 120 days
with only minor deviations; the syngeneic controls still exhibited
pretransplant frequencies (data not shown). In [BALB/c B6] BMT,
both proportional Ly49A+ and
Ly49C/I+ NK cell subsets of the total
donor/recipient DX5+ NK cell populations were
still significantly lowered (Fig. 5). The recipient
Ly49A+/NK1.1+ B6 NK cell
proportions in [BALB/c B6] BMT were less reduced (7.5 ±
6%, data not shown) than the DX5+ subset
(1.2 ± 1%, Fig. 5); the
Ly49C/I+/NK1.1+ recipient
subset was more reduced (9.5 ± 1%, data not shown) than the
total donor/recipient
Ly49C/I+/DX5+ NK cell
population (12.7% ± 6, Fig. 5), indicating a maintained
down-regulation in the presence of the cognate
H-2d donor ligand. After [B6 BALB/c] BMT,
the Ly49A+/DX5+ subset
(Fig. 6) was still significantly reduced as well as the donor-derived
Ly49A+/NK1.1+ subset
(3.4%, Fig. 7) (5.6 ± 2.3%, data
not shown) in contrast to the syngeneic controls (14.7 ± 4.1%;
data not shown). The only significant difference occurring after 120
days compared with day 55 was the reduction of the previously
normalized Ly49C/I+/DX5+
total donor/recipient NK cell proportion after allogeneic [B6
BALB/c] BMT compared with day 0 (Fig. 7) and in contrast to day 55
(Fig. 6). The donor-derived
Ly49C/I+/NK1.1+ subset was
reduced (24.3 ± 7.7%, data not shown) (24.6%, Fig. 7) in
contrast to the syngeneic controls (34.1 ± 3.3%).
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Double expression of Ly49A and Ly49C/I could only be examined on
B6 NK cells due to the staining specificity of mAb A1. Fifty-five days
after allogeneic [BALB/c B6] BMT, recipient B6 NK cells
expressing both receptors were proportionally diminished (3.4 ±
2.5%, Fig. 5). After [B6 BALB/c] BMT, double-expressing donor B6
NK cells were also significantly reduced (4.8 ± 2.6%, Fig. 6)
compared with day 0 (B6: 19 ± 6.9%, Figs. 5 and 6). Therefore
120 days after both allogeneic BMTs they were still significantly
reduced (Figs. 5 and 6). There was no difference compared with the
syngeneic [B6 B6] controls as they also exhibited significantly
reduced proportions 55 and 120 days after BMT (6.7 ± 3% and
8.1 ± 1%, respectively) (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10–20% and 80–90%,
respectively, of peripheral NK cells after 40–55 days, remaining
stable after 120 days. Because the life span of a murine NK cell is
<1 wk (39), the recipient NK cells presumably consisted
of radioresistant mature as well as newly developing NK cells despite
the myeloablative regimen. The degree of donor chimerism and the
survival rates were influenced by the number of grafted cells because
transplantation of more graft cells and magnetic depletion of T cells
in later experiments increased both (data not shown). Histology and
blood tests provided evidence that failure of engraftment was a major
cause of death rather than GvHD (data not shown). The initially changing MHC class I environment during establishment of chimerism especially challenges the Ly49 receptor repertoire formation of the developing donor and recipient NK cells during the first 8 wk after BMT. Low expression of MHC class I could be indicative of an immature population of NK cells compared with high expression levels on mature NK cells, previously described for GM-CFU cells (40). Whether these different levels of MHC class I on NK cells influence their expression of Ly49 receptors is a question for further investigations.
During normal ontogeny after birth splenic Ly49+ NK cells gradually rise to adult numbers during the first 6–8 wk of life (41). To assure that the alterations of Ly49+ subset frequencies we observed after allogeneic BMT were not merely a result of normal ontogeneic variations of developing NK cells, we performed syngeneic controls. Pretransplant frequencies of Ly49A- and Ly49C/I-expressing NK cells were similar to other studies (42).
It has to be considered that the initial challenge for the donor NK cells entering the new MHC environment is different to later time points when the donor itself constitutes the majority of BM cells and peripheral BM-derived cells. Even then the recipient’s MHC class I expression continues to exert influence. The recipient NK cells initially face a known MHC environment but encounter an increasing number of donor cells expressing non-self MHC class I molecules. Forty to 50 days after BMT, acute rejection was most likely overcome in the surviving mice. To keep the host alive, recipient and donor NK cells must have adapted their Ly49 receptor-mediated recognition pattern by then. We hypothesize that regulation of Ly49+ subset frequencies that recognize non-self MHC class I molecules is required of donor and host NK cells alike in a chimeric host.
With the present results it was shown that regulation of Ly49+ subset frequencies is important in allogeneic BMT extending the observations of other groups that focused on expression levels in vitro (43) or in vivo after BMT (44). Our findings provide evidence that the numbers of NK cells expressing a certain Ly49 receptor are influenced by host MHC class I molecules. This influence was first described by Held et al. (33). They found higher numbers of Ly49A+ NK cells in ß2m- than in ß2m+ mice. In the present study, we proved a regulatory effect of new MHC ligands in allogeneic BM chimeras. Above all, reductions of NK cell subset frequencies were observed with the introduction of new H-2b or H-2d ligands after allogeneic BMT. This result would be the inverse effect to the ß2m- mice because of more H-2 alleles being presented in the chimeric host. The reductions observed were confined to Ly49+ subsets within a total peripheral NK cell population of normal or even increased size.
Furthermore, the period of time crucial for the regulation was
determined. The reductions occurred early after 15–20 days post-BMT
in the total and donor NK cell populations. In contrast they tended to
be established later after 25–45 days in the recipient subsets and
were most evident after 55 days. Therefore the first 8 wk post-BMT is
the critical period of time for NK cells to adapt their Ly49 expression
to ensure tolerance. We assume that stability of new expression
patterns depended on stability of chimerism that was reached 50–60
days after BMT. Ly49+ subset repertoires of
mature NK cells are thought to adapt continuously (15). On
the one hand, we observed rather stable frequencies of
Ly49A+, Ly49C/I+, and
Ly49A+/Ly49C/I+ NK cell
subsets over 120 days, on the other hand minor changes of chimerism
(10%) and of subset frequencies, e.g., the down-regulation of the
previously normalized
Ly49C/I+/DX5+ subset 120
days after [B6 BALB/c] BMT. The delayed reduction of the recipient
Ly49AB6 and Ly49C/I B6
subsets might be explained by the initially lower but then growing
impact of graft MHC reaching full BM extension 40–50 days after BMT.
In contrast, the earlier reduction of the donor
Ly49AB6 and Ly49C/I B6 NK
cell proportions 15–20 days after [B6 BALB/c] BMT could result
from the greater impact of the external non-self recipient BALB/c
environment from the beginning of engraftment, including the decreasing
irradiated hematopoietic cells as well as nonhematopoietic elements.
Accordingly, the strong decrease of the total
Ly49C/I+/DX5+ subset on day
15 after [BALB/c B6] BMT also implied a reduction of the donor
Ly49CBALB/c subset.
To interpret these results we refer to the calibration model describing down-regulating effects on Ly49 expression levels by MHC-ligands in the surrounding external environment (14) and as recently proposed also for internal MHC expressed by NK cells themselves (15, 22). In the present study, it was investigated whether this model also applied to calibration of NK cell frequencies and whether the influence by external ligands was different when expressed in the whole microenvironment of the recipient or only by donor BM cells and BM-derived donor cells. In our chimeric models, both recipient and donor MHC class I ligands obviously had a down-regulating influence on Ly49+ NK cell population sizes as discussed below.
First we examined the influence of a new but cognate host environment
on the expression frequency of Ly49A
B6/H-2b donor-derived NK
cells in [B6 BALB/c] BMT. The donor Ly49AB6
NK cell proportions were significantly reduced.
Ly49AB6 NK cells recognize
H-2Dd and H-2Dk ligands
(38). Thus they recognized the external non-self
H-2Dd ligand expressed by the recipient BALB/c
environment, a new but cognate ligand, and were therefore
down-regulated. Then we examined the influence of donor BM cells and
BM-derived donor cells on recipient Ly49AB6 NK
cells in [BALB/c B6] BMT. We also observed a reduction of the
recipient Ly49AB6 NK cells. They recognized the
external non-self donor H-2Dd ligand expressed by
the graft in the chimeric BM and by BM-derived donor cells in the
periphery, a new but cognate ligand, and were therefore down-regulated
as well.
As for Ly49C/I we examined the modulation of donor
Ly49CBALB/c/H-2 d NK cell
proportions in the H-2 b host environment after
[BALB/c B6] BMT and found a significant reduction.
Ly49CBALB/c recognizes the ligands
H-2Kb, H-2Kd and
H-2Dd. Thus the donor
Ly49CBALB/c NK cells in [BALB/c B6] BMT can
interact with the cognate H-2Kb ligand of the
host environment resulting in down-regulation. Vice versa we examined
the modulation of the recipient Ly49CBALB/c
subset by the H-2b ligand. Here also, a decrease
of the recipient Ly49CBALB/c subset was observed
50 days after [B6 BALB/c] BMT and was implied in the reduced mean
total Ly49C/I+/DX5+ subset
frequency after 120 days. This is likely to be due to interaction with
the donor-derived cognate H-2Kb ligand. In
contrast to the donor Ly49CBALB/c NK cells, donor
Ly49C/IB6/H-2b NK cells
develop in a new and noncognate H-2d host
environment after the [B6 BALB/c] BMT because they only recognize
H-2Kb. As they did not recognize the external
non-self H-2d ligand they were not
down-regulated. Another important aspect is that this increase of
Ly49CB6 NK cells could also foster the
engraftment of their own BM cells as described in vitro by Murphy et
al. (18) for syngeneic Ly49C+ donor
NK cells compared with Ly49C- NK cells. Herein
may lie an explanation for the repeatedly better survival rate of
H-2b H-2d bone chimeras
in contrast to H-2d
H-2b chimeras. Yet, an increase of the
Ly49I+ proportion alone along with a decreased
Ly49C+ subset has to be taken into account. The
analysis of the double-expressing Ly49A+ and
Ly49C/I+ NK cells revealed a significant decrease
55 and 120 days after both allogeneic and syngeneic BMTs. The
mechanisms of this similar alteration remain unclear at present.
Regarding the maintenance of the altered expression patterns 120 days
after [B6 BALB/c] BMT compared with 55 days, the previously
normalized total donor/recipient
Ly49C/I+/DX5+ subset
frequency had significantly declined implying a reduction also of the
Ly49CB6 donor subset. This reduction might
indicate a down-regulating influence of the donor-derived external
and/or even internal self H-2b ligands which is
presumably not strong enough within the first 8 wk but increases with
the growing graft and leads to more interaction of donor
Ly49CB6 NK cells with H-2Kb
on surrounding cells. This reduction also occurred much later in
contrast to the early reduction of the recipient
Ly49C/IB6 NK cells after transplantation of the
H-2d noncognate ligand in [BALB/c B6] BMT.
There, the recipient Ly49C/IB6 NK cells were
reduced in the presence of the non-self noncognate donor
H-2d ligand and the self cognate
H-2Kb ligand. This might be explained by the
initially greater amount of surrounding self ligand-expressing cells in
contrast to the late reduction of the donor
Ly49CB6 subset above. This observation is also
consistent with the delayed reduction of the recipient
Ly49AB6 NK cells.
We cannot clearly define the influence of the noncognate donor
H-2d ligand and the self cognate
H-2Kb ligand, but postulate that the
Ly49CB6 recipient NK cells were only reduced
because 1) they express the cognate ligand themselves, and/or 2) they
recognize it on surrounding cells expressing the self cognate
H-2Kb ligand, and 3) this effect only occurs in
chimeric but not syngeneically transplanted hosts. This result might
provide the first in vivo observation of internal calibration with
respect to the frequency of Ly49-expressing NK cells or a regulatory
effect of self external ligands only in chimeric but not syngeneic
hosts. A down-regulatory effect of the noncognate donor
H-2d ligand is unlikely because 1) it is simply
not recognized and 2) our findings primarily showed that reduction of
Ly49A+ and Ly49C/I+ subsets
occurred upon interaction with cognate ligands. The opposite
normalization of the donor Ly49CB6 NK cell
frequency in the noncognate H-2d host confirmed
this assumption. The influence of Ly49IB6 that
does not bind to H-2d or
H-2b target cells or any other known ligand
(36, 38) remains unclear. Therefore our observations
provide further evidence in line with a recent study (34)
that self-MHC ligands also down-regulate Ly49+
subsets of MHC-identical NK cells only in allogeneic but not in
syngeneic hosts. Manilay et al. (34) showed that numbers
of donor and host Ly49A+ NK cells do not change
in allogeneic H-2d H-2b
BM chimeras and donor Ly49CBALB/C NK cells were
first increased then the same as those of nontransplanted controls
whereas recipient NK cells were decreased after 7 wk.
We conclude from our results that external cognate non-self MHC class I ligands down-regulate the frequencies of developing MHC-different Ly49+ NK cells in a chimeric host. Furthermore we assume that only in a chimeric host cognate self external ligands are also effective in the absence of a cognate non-self ligand. We also confirm that alterations are determined by the amount of ligands dependent on population sizes. During their development in the same host MHC class I-different recipient and donor NK cells adapted different Ly49 expression patterns within the first 55 days but after 120 days they both had acquired reduced Ly49+ subset frequencies. Therefore the frequencies between the Ly49A and Ly49C/I subsets differ, but are balanced between the strains as in nontransplanted MHC class I-different mice. In conclusion these results suggest a bilateral process of adaptation in mixed BM chimeras and strengthen the notion that maintenance of self-tolerance by NK cells in a changing, heterogeneous MHC environment is important for successful engraftment.
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2 Address correspondence and reprint requests to Dr. Reinhold E. Schmidt, Department of Clinical Immunology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail address:
3 Abbreviations used in this paper: BM, bone marrow; ß2m, ß2-microglobulin; KIR, killing inhibitory receptor; BMT, BM transplantation; GvHD, graft-vs-host disease; FCM, flow cytometry.
Received for publication June 15, 1999. Accepted for publication September 16, 1999.
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