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Institut National de la Santé et de la Recherche Médicale Unité 563, Centre de Physiopathologie de Toulouse Purpan, Institut Claude de Préval, Hôpital Purpan, Toulouse, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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rather than type 2 cytokines. Turning host NK cells
off was sufficient to restore strong alloreactive CD4 T cell priming
and Th2 cell development. Similar results were obtained by analyzing
the effect of NK cell activation on CD4 T cell responses to skin
allografts. However, despite the dramatic effect of NK cells on
alloreactive Th1/Th2 cell development, the kinetics of skin graft
rejection were not affected. Thus, Th2 differentiation is a major
pathway of alloreactive CD4 T cell development during solid organ
transplant rejection, as long as host NK and CD8 T cells are not
activated. We propose the hypothesis that MHC class I-driven
interactions between donor DCs and host NK cells or CD8 T cells might
result in DC-carried signals controlling the dynamics of alloreactive
CD4 T cell priming and polarization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although much progress has been made in understanding the role of NK cells in bone marrow transplantation, little is known about their function in CD4 T cell-mediated allogeneic transplant rejection (4, 5, 6, 7). For instance, in fully allogeneic combinations, it is likely that in addition to CD4 and CD8 T lymphocytes, NK cells can be activated by donor-derived APCs. Among donor APCs, tissue-resident dendritic cells (DCs)3 are likely to migrate from the graft to secondary lymphoid organs where priming of alloreactive T cells can occur initiating transplant rejection (8, 9). How NK cells can interfere with donor-derived DC and whether these interactions can modulate allospecific T cell development in vivo is presently not known. We have recently analyzed the development of alloreactive CD4 T cells in the absence of CD8 T cell activation in vivo. We showed that while immunization of adult mice with semiallogeneic splenocytes induces the differentiation of donor-specific CD4 T cells toward the Th1 phenotype, responses strongly polarized toward the Th2 type occurred in CD8-deficient mice. This led us to conclude that in the absence of CD8 T cell priming and whatever the genetic background of the host, the default response of alloreactive CD4 T cells is a sustained production of Th2-type cytokines (10).
In this study, we investigate the role of NK cell activation on the
development of alloreactive CD4 T cells in vivo in the absence of CD8 T
cell activation. The experimental model we have chosen involved priming
of the CD8-deficient parental C57BL/6 (B6) or BALB/c strains with
either semiallogeneic (BALB/c x B6) F1 (CB6
F1) or fully allogeneic APCs. Semiallogeneic APCs
that expressed both parental MHC products were used instead of fully
allogeneic cells to avoid NK cell activation (10).
Although immunization of CD8-deficient mice with semiallogeneic APCs
induced strong alloreactive CD4 T cell priming and Th2 cell
development, injection of allogeneic APCs resulted in reduced
allospecific CD4 T cell responses and in the selective expansion of
IFN--producing cells. Ab-mediated NK cell depletion before
immunization resulted in dramatic expansion of alloreactive Th cells
secreting high levels of IL-4 and IL-10 similar to the responses
observed in 
2-microglobulin
(2m) and CD8 double knockout mice where NK
cell activity is impaired (11, 12, 13). Interestingly,
grafting CD8-deficient mice with semiallogeneic skin also induced
marked allospecific Th2 cell priming; whereas rejection of allogeneic
grafts was associated with the selective development of Th1 cells. In
this latter combination, turning NK cells off resulted in a skewing of
the alloreactive CD4 T cell response to the Th2 pathway without
affecting the kinetics of skin graft rejection. Taken together, our
data demonstrate that recognition of donor APCs by host NK cells
strongly affects the magnitude of allospecific Th cell responses as
well as their cytokine secretion profiles in various donor/recipient
strain combinations.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c (H-2d), C57BL/6 (B6)
(H-2b), and (BALB/c x C57BL/6)
F1 (CB6 F1) mice were
purchased from Center d’Elevage R. Janvier (Le Genest St Isle,
France). H-2b mice with disrupted
2m genes were back-crossed to BALB/c mice as
previously described (14).
2m-/- mice on BALB/c
or B6 background were used after 10 back-crosses.
CD8-/-,
2m-/- mice on the B6
background were initially obtained from the Center National de la
Recherche Scientifique (Centre de Développement des Techniques
Avancées, Orléans, France) and were used to generate
double-deficient CD8-/- and
2m-/- B6 mice.
H-2b mice with disrupted CD8 genes were
back-crossed eight times to BALB/c mice in our own animal facility.
BALB/c 2m-/-, BALB/c
CD8-/-, B6
2m-/-, B6
CD8-/-, B6
CD8-/-2m-/-
and CB6 F1
2m-/- mice were bred
and maintained in our specific pathogen-free animal facility. Adult
mice (8–12 wk old) were immunized s.c. in the hind footpads with
50 x 106 irradiated (2400 rad) spleen cells
(SCs) or 0.5 x 106 bone
marrow-derived DCs (BM-DCs) from normal or
2m-deficient BALB/c or B6 or CB6
F1 mice. Six days after immunization, the
draining popliteal and inguineal lymph nodes were removed and further
processed as described below.
In vivo mAb treatment
For in vivo NK cell depletion in B6 mice, the anti-NK cells PK136 mouse IgG2a mAb (HB 191; American Type Culture Collection, Manassas, VA) was purified from ascites fluid by caprylic acid precipitation. For in vivo NK depletion, mice were injected i.v. with 200 µg PK136 mAb or with 25 µl anti-asialo GM-1 in 200 µl PBS 2 days prior to immunization, followed by two injections of 100 µg PK136 mAb or 25 µl anti-asialo-GM-1 in 200 µl PBS the day of immunization (day 0) and 2 days later. Anti-rat transferrin receptor OX26 mouse IgG2a was used as isotype control for PK136 anti-NK treatment in B6 mice and was kindly provided by Dr A. Saoudi (Institut National de la Santé et de la Recherche Médicale, Toulouse, France). In BALB/c mice, NK depletion was obtained by the administration of anti-asialo-GM-1 rabbit serum (Wako Chemicals, Richmond, VA). Rabbit serum was precipitated with 50% ammonium sulfate, dialyzed in PBS then adjusted at the same concentration as anti-asialo GM-1, and used as control for anti-NK treatment in BALB/c mice.
BM-DCs
BM-DC were generated as previously described (15). Briefly, bone marrow cells were cultured in complete medium supplemented with GM-CSF (Sigma-Aldrich, St. Louis, MO) at 2 x 105 cells/ml in a bacteriological petri dish (Greiner, Nurtingen, Germany). Complete medium was RPMI 1640 (Life Technologies, Cergy Pontoise, France) supplemented with 10% FCS (Life Technologies), 1% pyruvate, 1% nonessential amino acids, 1% L-glutamine, 50 µM 2-ME, and 50 µg/ml gentamicin (Sigma-Aldrich). The DC preparation was used at day 10 of culture. Flow cytometric analysis using N418 anti-CD11c mAb (HB 224; ATCC) revealed a purity >95% of CD11c+ cells, while Gr1-positive cells detected using anti-Ly6G mAb (BD PharMingen, San Diego, CA) were <5%.
T cell assays
For cytokine production analysis, CD8-depleted lymph node cells
(LNCs) from mice immunized with allogeneic SCs were cultured at 3
x 105 cells/well in 96-well culture plates
(Costar, Cambridge, MA) in the presence of various concentrations of
irradiated SCs. For removal of CD8 cells, LNC were incubated with KT1.5
mAb (16) culture supernatant, washed, and subsequently
incubated with sheep anti-rat IgG M-450 Dynabeads (Dynal Biotech,
Great Neck, NY) previously adsorbed with 10% normal mouse serum.
CD8-positive cells were then selectively depleted with a magnet
(BioSource International, Camarillo, CA). A similar procedure was used
for CD4 T cell enrichment by incubating LNC with anti-B220 RA3-3A1
(TIB 146; ATCC), anti-CD8 KT1.5, anti-class II M5/114 (TIB 120;
ATCC), and anti-CD11b (TIB 128; ATCC) mAb. Cells were cultured in
HL-1 synthetic medium (Hycor, Irvine, CA) supplemented with 2 mM
L-glutamine and 50 µg/ml gentamicin (Sigma-Aldrich).
Cultures were incubated for 3 days in a humidified atmosphere of 5%
CO2 in air. Supernatants from replicate cultures
were collected after 72 h and pooled for cytokine analysis.
IFN-, IL-4, IL-5, and IL-10 were quantified by two sites of sandwich
ELISA as previously described (10). For T cell
proliferation assays, cell cultures were pulsed 16 h with 1 µCi
[ 3H]TdR (40 Ci/nmol; Radiochemical Center,
Amersham, U.K.) before harvesting on glass fiber filter. Incorporation
of [3H]TdR was measured by direct counting
using an automated -plate counter (Matrix 9600; Packard Instrument,
Meriden, CT).
Flow cytometric analysis of intracellular cytokine synthesis
LNC were cultured with allogeneic-irradiated (2400 rad) SCs from
B6 or BALB/c or CB6 F1 mice as indicated above.
After 72 h of culture, cells were harvested, washed, and cultured
for an additional 72 h in complete medium. After Ficoll
separation, living cells were collected, resuspended at
106/ml, and stimulated with PMA (50 ng/ml;
Sigma-Aldrich) plus ionomycin (0.5 µg/ml; Sigma-Aldrich) for 4
h. Two hours before cell harvest, brefeldin A (Sigma-Aldrich) was added
at a concentration of 10 µg/ml. Cells were harvested, washed in the
presence of brefeldin A, and stained using biotinylated anti-CD4
mAb (BD PharMingen), followed by streptavidin-CyChrome (BD PharMingen).
Labeled cells were then fixed with 2% paraformaldehyde (Fluka Chemie,
Buchs, Switzerland). Intracytoplasmic staining for IFN-, IL-4, IL-5,
and IL-10 were performed as described previously (10).
Data were collected on 20,000 CD4+ cells on a XL
Coulter cytometer (Beckman Coulter France S.A., Roissy, France)
and analyzed using the CellQuest software (BD Biosciences, Mountain
View, CA).
Skin grafting
Skin grafts 
1 cm in diameter were prepared from tails of
female CB6 F1, B6, or BALB/c mice and grafted
onto the flank of recipient mice. Petroleum gauze was placed over the
graft and sticking plaster was applied around the trunk. After 7 days,
bandages were removed and the grafts monitored daily. Grafts were
considered rejected when complete epithelial breakdown had
occurred.
Statistical analysis
Results are expressed as the mean ± SD, and statistical differences between variables were evaluated by the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that priming of alloreactive CD4 T cells
with semiallogeneic APCs results in the development of effector Th
cells producing large amounts of type 2 cytokines in the absence of CD8
T cell activation (10). This is illustrated in Fig. 1
, where immunization of
2m-/-BALB/c mice with
semiallogeneic
2m-/--irradiated
splenocytes resulted in the generation of
I-Ab-reactive CD4 T cells producing high levels
of IL-4 and IL-10 in agreement with our previous work
(17). This was confirmed by the analysis of intracellular
cytokine synthesis in the same T cell populations after 6 days of in
vitro stimulation with CB6 F1 APCs (Fig. 2C). Strong expansion of IL-4-
or IL-10-producing cells was observed, and accounted for the majority
of effector CD4 T cells in this combination. In contrast, the frequency
of CD4 T cells that produced IFN- was <10%. Conversely,
immunization of BALB/c mice with CB6 F1 APCs
induced the development of IAb-reactive Th1
cells, producing mainly IFN- (38%) but little type 2 cytokines
(Fig. 2A). We have previously shown that the difference in
alloreactive CD4 T cell phenotype acquisition between these two
combinations was due to the in vitro activation of MHC class I-specific
CD8 T cells (10). Because MHC class I deficiency in
hemopoietic cells has been shown to promote NK cell activation, we next
evaluated the effect of priming BALB/c mice with
2m-deficient CB6 F1
APCs. As shown in Fig. 1, priming BALB/c mice with MHC class
I-deficient CB6 F1 APCs induces
H-2b-reactive CD4 T cells able to proliferate and
to secrete IFN-, but little if any type 2 cytokines upon in vitro
restimulation. Indeed, most of the alloreactive effector CD4 T cells
that develop in this combination exhibited a Th1-phenotype as shown by
intracellular cytokine staining (Fig. 2B). To demonstrate
directly that NK cell activation was responsible for this effect,
recipient mice were pretreated with asialo-GM-1 antiserum. As shown in
Fig. 3, NK cell depletion resulted in the
restoration of IL-4 and IL-10 production by
H-2b-reactive CD4 T cells. The T cell
proliferative response, as well as IFN- synthesis were also
up-regulated in anti-asialo-GM-1-treated mice (Fig. 3). Thus,
impaired MHC class I expression by donor APCs dramatically affects the
magnitude and the phenotype of the allospecific CD4 T cell response due
to the absence of negative regulation of host NK cells.
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NK cell-mediated bone marrow rejection can be caused by the
complete lack of expression of MHC class I molecules, but also by the
lack of self MHC class I molecules on donor cells (2, 18)
albeit less efficiently (11, 12). To evaluate the effect
of NK cell activation on alloreactive CD4 T cell development, BM-DC
from either fully allogeneic B6 or semiallogeneic CB6
F1 mice were used to immunize BALB/c
CD8-/- recipients. Draining lymph nodes were
harvested 6 days later and LNC restimulated in vitro with titrated
amounts of semiallogeneic irradiated splenocytes (Fig. 4). In agreement with our previous
experiments (10), priming of CD8-deficient BALB/c mice
with semiallogeneic CB6 F1 BM-DCs induced a
vigorous expansion of memory/effector CD4 T cells secreting large
amounts of type 2 cytokines in response to allogeneic MHC class II
products (Fig. 4, A and B). Similar results were
obtained using allogeneic B6 SC as APCs (data not shown). In contrast,
although a proliferative response could be induced in CD8-deficient
BALB/c mice primed with B6 BM-DCs (Fig. 4A), these
B6-specific CD4 T cells secreted less IFN- in primary culture and no
type 2 cytokines (Fig. 4B). To test whether this effect was
due to NK cell activation as a consequence of the lack of expression of
self MHC class I molecules, mice were pretreated with asialo-GM-1
antiserum. The B6-specific proliferative response was slightly
up-regulated in NK-depleted mice as compared with control serum-treated
and untreated CD8-deficient BALB/c recipients (Fig. 4A).
Interestingly, in vivo elimination of NK cells led to the expansion of
allospecific CD4 T cells producing high amounts of IL-4 and IL-10 in
BALB/c CD8-/- mice immunized with fully
allogeneic B6 APCs (Fig. 4B). IFN- synthesis was also
up-regulated in mice treated with anti-asialo GM-1 Abs (Fig. 4B). Because both type 1 and 2 cytokines were increased by
turning NK cells off, we analyzed the frequency of IL-4- and
IFN--producing cells. As shown in Fig. 4C, NK cell
depletion resulted in a dramatic increase in effector/memory Th2 cell
expansion producing IL-4 but no IFN- (40–60%). The remaining
effector T cells were composed of Th1 (2–6%
IFN-pos) and Th0 (3–7%
IL-4posIFN-pos)
lymphocytes. This pattern of cytokine-producing cells was similar to
the one observed in CD8-deficient BALB/c mice primed with CB6
F1 APCs (Fig. 4C). Although T cell
proliferative responses when measured in the fully allogeneic
combination were associated with IFN- synthesis (Fig. 4, A and B), the yield of allospecific CD4 T cells
after 6 days of primary culture was very low (<0.2 x
106 cells for 106
input cells) precluding further analysis (data not shown). However, in
some experiments where a sufficient number of T cells could be obtained
from this combination, the response was dominated by IFN--producing
cells with a frequency of 20% (data not shown).
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Experiments described so far were performed in BALB/c mice using
anti-asialo GM-1 Abs for NK cell depletion, which can also damage
macrophages (19) or T cells (20). Therefore,
we tested whether our observations were also valid in B6 mice in which
the method of NK cell depletion using the anti-NK1.1 mAb PK136 is
well established (21). To examine the allogeneic CD4 T
cell response in the absence of CD8 T cell activation, we used
CD8-deficient B6 mice as recipients. We compared two combinations where
mice were injected with BM-DCs from either semiallogeneic CB6
F1 or allogeneic BALB/c mice. Unlike BALB/c
BM-DCs, CB6 F1 BM-DCs express MHC class I
molecules of H2b haplotype; and therefore, are
able to inhibit NK cell activation. As shown in Fig. 5, immunization of CD8-deficient B6 mice
with allogeneic BALB/c BM-DCs resulted in a low lymphocyte recruitment
in the draining lymph nodes (Fig. 5A). Upon allogeneic
stimulation with APCs expressing H-2d class II
molecules, LNC failed to proliferate (data not shown) and did not
produce cytokines (Fig. 5B). In contrast, immunization of
CD8-deficient B6 mice with semiallogeneic CB6 F1
APCs resulted in a strong expansion of
H-2d-specific CD4 T cells that produce, in
addition to IFN-, high levels of type 2 cytokines (Fig. 5, B and C), in agreement with our previous
observations (10).
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2m-deficient mice have
been shown to have an impaired NK activity due to the lack of
expression of MHC class I molecules (11, 12), CD4 T cell
priming in response to fully allogeneic APCs was also tested in CD8 and
2m double knockout B6 mice. As shown in Fig. 6A in agreement with data in
Fig. 5
, CD8-deficient B6 mice injected with BALB/c BM-DCs failed to
develop a significant allospecific CD4 T cell response. Lack of T cell
priming was also observed in B6 CD8-/- mice
treated with an irrelevant isotype-matched control mAb (data not
shown). In striking contrast, in NK-cell depleted B6
CD8-/- mice, a strong T cell proliferation and
cytokine production following in vitro allogeneic restimulation was
observed. Similar CD4 T cell responses were recorded in NK-depleted B6
CD8-/- mice and in
2m-deficient B6 CD8-/-
mice (Fig. 6A) consistent with an inhibitory effect of NK
cells on alloreactive T cell priming. To define the cytokine production
profile of allospecific CD4 T lymphocytes, expanded T cells were
stained intracellularly for IL-4 and IFN- after PMA/ionomycin
stimulation. Data shown in Fig. 6B illustrate that
allospecific CD4 T cells primed in the absence of NK cell activation,
either following Ab depletion (Fig. 6B, upper
panel) or in mice with impaired NK cell activity (Fig. 6B, lower panel), exhibited strikingly similar
profiles of cytokine-producing cells. The response was dominated by
IL-4-producing cells (up to 30% of CD4 T cells), of which one-third
also secreted IFN-. The frequency of
IFN-pos T cells was 10% of CD4 T cells.
Similar results were obtained when B6 CD8-/-
mice were primed with semiallogeneic F1 APCs
(Fig. 5C). Because it has been reported that syngeneic DC
could be killed efficiently by autologous NK cells (22),
and that there is a bidirectional cross-talk between DC and NK cells
(23), we tested the effect of NK cell depletion in mice
immunized with semiallogeneic DCs. Data in Fig. 7 show that in this combination where NK
cells are silenced by the expression of syngeneic MHC class I
molecules, NK cell depletion had no significant effect on alloreactive
CD4 T cell activation and differentiation. Thus, in agreement with our
previous observations in the BALB/c genetic background, inhibition of
NK cell activation in B6 recipients immunized with fully allogeneic DCs
enhances allospecific CD4 T cell priming and promotes Th2 cell
development.
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We next determined whether NK cell activation in the absence of
self-MHC class I expression can also control allospecific CD4 T cell
responses induced by tissue transplantation. To this end, we performed
skin graft on CD8-deficient BALB/c recipients in combinations in which
NK cells were activated (B6 donor) or not (CB6 F1
donor). As shown in Fig. 8A,
the outcome of CD4 T cell-mediated graft rejection was similar in
CD8-deficient recipient grafted with either a semi or fully allogeneic
skin, and occurred between days 8 and 11. We then analyzed the
polarization of allospecific CD4 T cells from lymph nodes draining the
graft before and during the rejection process. As shown in Fig. 8B, CD4 T cells from CD8-deficient BALB/c mice grafted with
CB6 F1 skin produced large amounts of IL-4 in
addition to IFN- upon restimulation with CB6
F1 APCs at both time points tested. In individual
mice, the level of IL-4 synthesis was inversely correlated to IFN-
secretion, indicating that strong Th2 polarization had occurred in CB6
F1-grafted recipients. In contrast, while IL-4
production was low or undetectable in recipient mice bearing fully
allogeneic graft, the allospecific CD4 T cell response was
characterized by the selective development of IFN--producing
cells.
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-producing T cells
(Fig. 9A). In contrast, NK
cell depletion in vivo resulted in enhanced IL-4 and decreased IFN-
production by T cells from the lymph nodes draining the BALB/c graft
(Fig. 9A). Interestingly, a similar skewing of the
H-2d-specific T cell response to the Th2
phenotype was observed in 2m- and
CD8-deficient B6 mice. In contrast to PK136-treated mice, those
injected with control mAb exhibited a Th1-polarized allospecific T cell
response. Finally, although the character of alloreactive CD4 T cell
responses was dramatically affected by NK cell depletion, the kinetics
of allogeneic BALB/c skin graft rejection in PK136-treated B6
CD8-/- mice was not modified, demonstrating
that acute graft rejection can occur in the context of either a Th1- or
Th2-polarized immune response (Fig. 9B). Taken together,
these data demonstrate that donor DC-driven host NK cell activation
controls alloreactive Th cell development during skin graft
transplantation without affecting the kinetics of rejection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2m-deficient BALB/c mice were immunized with
2m-deficient semiallogeneic CB6
F1 (H-2bxd) APCs. In
contrast, immunization of wild-type (WT) BALB/c mice with CB6
F1 APCs resulted in the selective development of
H-2b-specific Th1 cells (10). The
absence of Th2 cell development was controlled by CD8 T cells, because
immunization of CD8-deficient parental strains with CB6
F1 APCs induced allospecific CD4 T cells
producing a large amount of Th2-type cytokines (10). We
initially thought of using CB6 F1 APCs to prime
alloreactive host CD4 T cells on the assumption that
F1 APCs expressed, in addition to allogeneic MHC
products, self MHC class I molecules that bind to inhibitory receptors
on host NK cells, thereby silencing inhibitory receptor-bearing cells.
We now provide direct evidence that NK cells, through their interaction
with allogeneic APCs, can quantitatively and qualitatively control
allospecific CD4 T responses in vivo. This was tested in different
combinations: 1) by injecting 2m-deficient
allogeneic APCs into normal mice, or 2) by immunizing CD8-deficient
mice with fully allogeneic APCs. In the first combination, host NK
cells were likely to be activated as a consequence of the lack of
expression of self MHC class I molecules on donor-derived APCs, thus
resulting in NK cell activation. In this combination, the allospecific
CD4 T cell response was impaired and led to the development of a
Th1-dominated immune response. Depletion of asialo-GM-1-positive cells
before immunization with 2m-deficient APCs
restored a strong alloreactive CD4 T cell response characterized by the
emergence of type 2 cytokine-producing cells. These data indicate that
NK cells can regulate CD4 T cell activation and differentiation in
vivo. This was further demonstrated in the second type of combination
where inhibitory receptors on NK cells cannot be engaged by allogenic
donor APCs that unlike CB6 F1 APCs do not express syngeneic
MHC class I molecules. Again, NK cell depletion was found to enhance
CD4 T cell priming and to unmask Th2 cell development in both
CD8-deficient B6 and BALB/c-recipient mice. Furthermore, we showed that
H-2d-specific Th2 responses of comparable
magnitude were generated in NK-depleted B6
CD8-/- mice and in B6
CD8-/-
2m-/- double-deficient
mice that have an impaired NK cell activity due to the lack of MHC
class I molecules (11, 12, 13). Thus, our data demonstrate
that in various donor/recipient combinations where NK cells are
activated, allospecific Th2 cell responses are abrogated or strongly
diminished with the development of effector Th cells producing
selectively IFN- but no type 2 cytokines. Inhibition of host NK
cells was sufficient to induce alloreactive CD4 T cell responses
dominated by type 2 cytokine-producing effector Th cells. This
mechanism was not restricted to the BALB/c background and occurred also
in B6 mice. However, in this latter strain, NK cell activation resulted
in a complete inhibition of alloreactive CD4 T cell priming when DCs
were used for immunization. In contrast, when skin grafts were applied
as stimulus, unipolar Th1 cell responses were selectively observed in
combinations where NK cells were activated in both BALB/c and B6
strains. For instance, in CD8-deficient BALB/c recipients, Th1
responsiveness was not significantly different between mice grafted
with either allogeneic or semiallogeneic skin. Conversely, strong
priming of IL-4-producing T cells was exclusively observed in the
semiallogeneic combination. The impact of NK cell activation on Th1/Th2
differentiation was further demonstrated by grafting CD8-deficient B6
mice with allogeneic BALB/c skin. In this combination, turning NK cells
off resulted in a decreased production of IFN- and an increased
synthesis of IL-4 by alloreactive CD4 T cells from the lymph nodes
draining the graft. Taken together, our data support the conclusion
that NK cell activation reduces alloreactive CD4 T cell priming and
thus, the subsequent development of both subsets of memory/effector T
lymphocytes with a major impact on Th2 cells. Indeed, IL-4-producing
cells expand exclusively when NK cells were turned off. In contrast,
when CD4 T cell responses develop in combinations where NK cells are
activated by donor-derived APCs, these Th cells exhibited a unipolar
Th1 profile.
Our study unveils a novel mechanism by which NK may regulate the
adaptive immune response directed against transplantation Ags in the
absence of appropriate interaction between inhibitory receptors on NK
cells and MHC class I molecules on donor APCs. These observations are
relevant to the clinical transplant situation because similar
conclusions could be drawn by analyzing CD4 T cell responses to
allograft transplantation. Allospecific CD4 T cells producing IL-4
could readily be detected only when CD8-deficient mice were grafted
with semi-identical skin. In this combination, NK cells were not
activated by donor-derived DCs because they could receive a silencing
signal via self MHC molecules. In contrast, rejection of fully
allogeneic skin grafts was associated with the selective development of
Th1 cells due to NK cell activation by donor-derived DCs. Indeed, we
showed that inhibition of NK cells during CD4 T cell-mediated
allogeneic skin graft rejection induced a skewing of the alloreactive T
cell response to the Th2 pathway. Therefore, the impact of NK cell
activation on the development of Th2 effector functions was similar
when skin grafts or BM-DCs were used as stimulus. This conclusion is in
agreement with the hypothesis that donor-derived APCs, e.g., DCs, play
a central role in inducing CD4 T cell-mediated allograft rejection
through direct presentation of donor allogeneic MHC class II molecules
to host Th lymphocytes in the draining lymph nodes (8, 9, 24). However, despite the dramatic effect of NK cells on
alloreactive Th1/Th2 cell development, there was no effect on the
kinetics of skin graft rejection. This is not surprising since it has
been shown by others that both Th1 and Th2 responses are capable of
causing acute transplant rejection with identical kinetics in recipient
mice receiving either cardiac (25) or skin
(26) grafts. Furthermore, subsequent studies have
established that rejections mediated by Th2 cells were characterized by
marked eosinophilic infiltration of skin and heart transplants
(27, 28). Finally, it has been reported by Le Moine et al.
(29) in a model of MHC class II disparate skin grafts
(B6bm12
B6) that IL-4, IL-5, and
eosinophils were critically involved not only in chronic but also in
acute (30, 31) skin graft rejection. These observations
are in agreement with our present study and support the conclusion that
alloreactive CD4 T cell development during solid organ transplant
rejection is strongly biased toward Th2 phenotype as long as host NK
and CD8 T cells are not activated.
DCs have been shown to play a crucial role in allorejection by migrating from the transplanted tissue to secondary lymphoid organs of the host where they can prime allospecific T cells and initiate graft rejection (8, 9). Accumulating evidence indicates that DCs are phenotypicaly heterogeneous, and represent a multilineage system of leukocytes with variable functions (32). These DC subsets appear to play a role in determining the specific cytokines secreted by Th cells. It has been hypothesized that DCs recruited in immune lymph node can be instructed by environmental stimuli to perform different functions (33, 34). It has been shown in humans that IL-12 is produced by DCs within a narrow time window so that only recently activated DCs can promote Th1 cell development (33, 35). At later time points, DCs become exhausted in their capacity to secrete various cytokines including IL-12, thereby favoring conditions for priming of Th2 responses, which are dependent on IL-4 production by responding T cells (33, 34, 35). Transient IL-12 production by DCs in vivo have also been documented in mice following systemic stimulation with microbial products (36). To explain our data, we hypothesize that in a situation where both the NK and CD8 T cell pathways are not operative, donor DCs may accumulate in immune lymph nodes, thereby favoring strong CD4 T cell priming and Th2 type responses. Thus, tissue resident NK cells and possibly also CD8 T cells present in the secondary lymphoid organs would inhibit the default Th2 differentiation of allospecific CD4 T cells by limiting the flux of incoming allogeneic DCs into the draining lymph nodes and/or by shortening DC half-life in situ. This may lead to incomplete kinetics of DC differentiation preventing allospecific CD4 T lymphocyte activation by a particular DC subset with Th2-prone capacity. This would explain why in these conditions, CD4 T lymphocytes selectively differentiate toward the Th1 phenotype.
It has recently been shown that inhibition of NK cells combined with CD28 costimulation blockade induced long-term survival of allogeneic vascularized cardiac grafts. In contrast, none of these treatments alone resulted in the acceptance of cardiac allografts. Thus, it has been suggested that NK cells may have a critical role in allorejection by providing help to T cells, such function being essential in the absence of CD28-mediated costimulation (37). Our data are contradictory to this hypothesis, and several reasons could explain this discrepancy. First, CD28-deficient mice have reduced humoral responses but normal cell-mediated immunity (38), indicating that CD4 T cells are more profoundly affected by CD28-B7 blockade than CD8 T lymphocytes. Indeed, it has been shown that blockade of CD28/B7 costimulation inhibits intestinal allograft rejection mediated by CD4+ but not CD8+ T cells (39). Furthermore, it has been shown that CD28-B7 costimulation plays an important role in generating the Th2 compartment (40, 41, 42). Altogether, this could explain why no Th2 cell priming was observed by turning NK cells off in CD28-deficient mice (37). Second, the CD4/CD8 ratio among graft infiltrating cells was strongly skewed toward CD8 T cells in CD28-deficient mice (37), suggesting that CD8 T cells appear to be the major effector T cell subset responsible for graft rejection. In contrast, it has been clearly established that rejection of skin and cardiac allografts in mice with functional CD28/B7 costimulation pathways was dependent upon CD4 T cells, whereas CD8 T cells were never necessary nor sufficient (43, 44). Therefore, the observation that NK cells in allogeneic graft might preferentially provide help to CD8 T cells might be a particularity of the CD28-/- model and is most likely not relevant to the normal recipient situation where rejection mainly involves CD4 T cells (43, 44).
In conclusion, our data strongly support the hypothesis that NK cells rather than delivering help to allospecific CD4 T cells may actually limit their activation and differentiation in vivo through their interaction with allogeneic APCs. Indeed, it has been recently shown that donor NK cells from transplanted allogeneic bone marrow are able to eliminate host APCs, preventing donor T cell activation and the consequent graft-vs-host reaction (45). Therefore, our present observations should be taken into consideration in situations where NK cell-inactivating strategies are proposed to improve transplantation tolerance of allogeneic organs (37). According to our data, inactivation of both CD8 T cells and NK cells would promote allospecific CD4 T cell priming and type 2 cytokine production, resulting in an alternative pathway of solid organ rejection involving eosinophils (27, 28, 31) rather than transplantation tolerance. We hypothesize that NK cells and/or CD8 T cells may limit the flux of graft-derived DCs and/or their kinetics of differentiation in immune lymph nodes, thereby preventing alloreactive CD4 T cell activation by a subset of terminally differentiated DCs displaying Th2-prone capacity. Current experiments are in progress to address this issue.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean-Charles Guéry, Institut National de la Santé et de la Recherche Médicale Unité 563, Hôpital Purpan, Place du Dr Baylac, 31059 Toulouse cedex, France. E-mail address: jean-charles.guery{at}toulouse.inserm.fr
3 Abbreviations used in this paper: DC, dendritic cell; 2m, 2-microglobulin; BM-DC, bone marrow-derived DC; LNC, lymph node cell; SC, spleen cell; WT, wild type.
Received for publication April 1, 2002. Accepted for publication July 10, 2002.
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