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T Cells and NK Cells Inhibit the Engraftment of Xenogeneic Rat Bone Marrow Cells and the Induction of Xenograft Tolerance in Mice1
Bone Marrow Transplantation Section, Transplantation Biology Research Center, Surgical Service, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-, and B6 TCR
-/- mice received
anti-CD4 and anti-CD8 mAbs, followed by 3 Gy of whole body
irradiation, 7 Gy of thymic irradiation, and transplantation of T
cell-depleted rat bone marrow cells. Anti-NK1.1 and anti-Thy1.2
mAbs were additionally administered to some groups. Increased rat
chimerism was observed in TCR-/- mice treated
with anti-CD4, anti-CD8, and anti-NK1.1 mAbs compared
with similarly treated TCR-/- mice. In
TCR-/- mice, but not in TCR -/- mice,
donor chimerism was increased by treatment with anti-Thy1.2 mAb,
indicating that
CD4-CD8-TCR
+Thy1.2+NK1.1-
cells ( T cells) are involved in the rejection of rat marrow. In
addition, chimerism was enhanced in both TCR-/- and
TCR-/- mice treated with anti-CD4, anti-CD8,
and anti-Thy1.2 mAbs by the addition of anti-NK1.1 mAb to the
conditioning regimen. Donor-specific skin graft prolongation was
enhanced by anti-Thy1.2 and anti-NK1.1 mAbs in
TCR-/- mice. Therefore, in addition to CD4 and CD8 T
cells, T cells and NK cells play a role in resisting engraftment
of rat marrow and the induction of xenograft tolerance in
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies in this laboratory have shown that in addition to host
pretreatment with anti-CD4 and anti-CD8 mAbs, achievement of
xenogeneic rat marrow engraftment in mice requires host pretreatment
with mAbs targeting Thy1.2+ and
NK1.1+ cells (4). As in the
allogeneic system, these mixed chimeras show donor-specific tolerance
in vitro (by MLR and cell-mediated lympholysis (CML) assays) and
long-term donor-specific skin graft tolerance in vivo, with rapid
rejection of nondonor third-party rat skin grafts (4, 8).
We have recently demonstrated a role for rat bone marrow-derived cells
in negative selection of mouse T cells, as seen by deletion of
V+ host T cells that recognize superantigen in
association with rat MHC class II, suggesting that tolerance occurs via
a deletional mechanism in these chimeras (8, 12).
In mice, pretreatment with anti-CD4 and anti-CD8 mAbs
effectively depletes host alloresistant cells, but not xenoresistant
cells. This result suggests that additional xenoresistant cell
populations in the mouse are NK1.1+ and/or
Thy1.2+, and CD4 and CD8 negative. The goal of
this study was to identify the
CD4-CD8- xenoresistant
mouse cells targeted by anti-NK1.1 and anti-Thy1.2 mAbs. The
NKR-P1 receptor NK1.1 is expressed on
CD4-CD8-Thy1.2-NK1.1+
cells (NK cells) as well as on T cells that express NK cell-associated
markers (T/NK cells) (13, 14, 15). Thy1.2 is expressed on
and
CD4-CD8-TCR+Thy1.2+NK1.1-
cells ( T cells), T/NK cells, and a small population of activated
NK cells (16, 17, 18, 19, 20, 21, 22). Therefore, the potential xenoresistant
cells that must be depleted to achieve engraftment in the xenogeneic
rat-to-mouse model may be NK cells, T/NK cells, or double-negative
T cells or T cells, which lack CD4 and CD8, and would not
be depleted by anti-CD4 and anti-CD8 mAb pretreatment
alone.
To distinguish the potential roles of the above cell populations in
resisting xenogeneic marrow engraftment, we compared the mAb
requirements to achieve successful engraftment in C57BL/6 (B6)
wild-type (wt) mice, B6 TCR-/- mice, and B6
TCR-/-mice. B6
TCR-/- mice lack T cells
(23) and B6 TCR-/- lack
T cells and most T/NK cells (24). We treated these mice
with varying combinations of the anti-CD4, -CD8, -NK1.1, and
-Thy1.2-depleting mAbs and transplanted them. We then evaluated
these transplanted animals for the ability to achieve mixed chimerism,
and for donor-specific skin graft tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female B6 (H-2b), B6
TCR-/- (H-2b), B6
TCR-/- (H-2b), BALB/c
(H-2d), A.SW (H-2s), and
SJL/J (H-2s) mice were purchased from The Jackson
Laboratory (Bar Harbor, ME). Female Fisher 344 (F344;
RT1l) and Wistar-Furth (WF;
RT1u) rats were purchased from Charles River
Breeding Laboratories (Wilmington, MA). All mice and rats were housed
in sterilized microisolator cages in which they received autoclaved,
acidified drinking water and autoclaved food, as described
(25). The recipients in the experiments were age matched
and were 6–10 wk old.
Nonmyeloablative regimen and BMT
Anti-CD4 and anti-CD8 mAbs were administered i.p. to B6 wt,
B6 TCR-/-, and B6
TCR-/- mice. In addition, some groups
received anti-Thy1.2 mAbs, anti-NK1.1 mAbs, or both. All
injections were given on days -6 and -1 (4). Each
injection consisted of 1 ml of PBS containing rat anti-mouse CD4
IgG2b mAb GK1.5 (1.76 mg), rat anti-mouse CD8 IgG2b mAb 2.43 (1.50
mg), and either rat anti-mouse Thy1.2 mAb 30-H12 (0.55 mg), murine
anti-NK1.1 mAb PK136 (0.15 mg), or both. On day 0, the recipients
were treated with 7 Gy local TI, and 3 Gy WBI as described
(4). Animals then received 60 x
106 F344 rat BMC that were T cell-depleted (TCD)
using anti-CD5 mAb R1-3B3 (26) and two cycles of
rabbit complement as described (4).
Detection of mixed chimerism
Peripheral whole blood from tail bleeds was lysed by hypotonic
shock, yielding white blood cells (WBC). WBC were analyzed by two-color
flow cytometry after staining with FITC-conjugated murine IgG1
anti-rat MHC class I mAb Ox-18 (PharMingen, San Diego, CA). Rat T
cells were detected using FITC-conjugated mouse anti-rat CD4 mAb
(W3/25) (Accurate Chemical and Scientific, Westbury, NY), and mouse
anti-rat CD8 mAb (Ox-8) (Harlan Bioproducts for Science,
Indianapolis, IN). FITC-conjugated and biotinylated mAb HOPC1 (mouse
IgG2a) plus PE-streptavidin were used as nonstaining irrelevant Abs.
Nonspecific FcR binding was blocked with 10 µl of undiluted
culture supernatant containing rat anti-mouse FcR mAb 2.4G2
(27). Cells were analyzed by two-color flow cytometry
using a FACScan (Becton Dickinson, Mountain View, CA). For calculation
of the percentage of donor cells, staining with control mAb was
subtracted from the percentage of cells staining with the appropriate
test mAb, in the same two-color dot plot region (28). The
net positive percentage of rat donor cells was calculated after
subtraction of staining with FITC-conjugated HOPC1 in the same dot plot
region. Cell lineages were identified using forward angle scatter (FSC)
and 90o light scatter (SSC) dot plots. WBC were
grouped into lymphocyte (FSC low and SSC low), granulocyte (FSC
intermediate and SSC high), and monocyte (FSC high and SSC low)
populations, and the percentage of Ox-18+ rat
donor cells was calculated for each individual cell population. Dead
cells were excluded by gating out low FSC/high propidium
iodide-retaining cells.
Skin grafting
Skin grafting was performed 103–106 days post-BMT as described previously (29). Full-thickness tail skin (1 cm; Ref. 2) was taken from donor rats and mice. Graft beds (1 cm2) were prepared on the right and left lateral thoracic walls of recipient mice. Grafts were secured with sutures and an adhesive bandage. The bandages were removed and the first observation was made on the seventh postoperative day, followed by inspection every day for the first month and two to three times a week thereafter. Animal care was in accordance with the American Association for the Accreditation of Laboratory Animal Care and institutional guidelines. All operations were performed under metofane anesthesia.
Statistical analysis
Statistical significance was determined using Student’s t test for comparison of means. A p value <0.05 was considered to be statistically significant. Skin graft survivals were analyzed using Kaplan-Meier plots.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mouse chimerism and tolerance in B6 wt
recipients
Xenogeneic rat chimerism in groups of wt B6 recipients was
determined by staining peripheral WBC with mAb that recognized rat MHC
class I (Ox-18). As is shown in Fig. 1
, mice that received pretreatment with anti-CD4 and -CD8 mAbs had the
lowest level of xenogeneic chimerism at wk 3 (mean 0.3%, range
0.0–0.7%) and wk 6 (mean 0.1%, range 0.0–0.3%). Adding
anti-Thy1.2, anti-NK1.1, or both to the conditioning regimen
increased the level of rat peripheral chimerism. The highest levels of
rat peripheral chimerism were found in the group that received
pretreatment with all four mAbs (mean 5.2% at wk 3 and 6.4% at wk 6),
which were significantly higher than those in the group receiving only
anti-CD4 and -CD8 (p < 0.01 for weeks 3
and 6), the group receiving anti-CD4, -CD8, and Thy1.2
(p < 0.001 for weeks 3 and 6), and the group
receiving anti-CD4, -CD8, and -NK1.1 (p <
0.05 for weeks 3 and 6). The group receiving anti-CD4, -CD8, and
-NK1.1 mAbs had significantly higher rat peripheral chimerism than the
group receiving anti-CD4 and -CD8 mAbs alone at wk 3
(p < 0.001) and at wk 6
(p < 0.05). The group receiving pretreatment
with anti-CD4, -CD8, and -Thy1.2 mAbs had significantly higher
levels of rat peripheral chimerism compared with the group receiving
anti-CD4 and -CD8 mAbs at 3 wk (p < 0.05),
but the differences did not achieve statistical significance at 6 wk.
Similar results were obtained for gated rat monocyte, granulocyte, and
lymphocyte lineages when analyzed separately (data not shown). Table I shows the levels of rat class
I+ cell mixed chimerism in groups of ratwt
chimeras in four separate experiments at 3 wk post-BMT.
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B6 wt BMT recipients were grafted with donor-type F344
skin grafts 10 wk following BMT to determine which groups of
conditioned animals were tolerant to donor Ags. Skin grafts were
followed for 72 days. The group receiving pretreatment with only
anti-CD4 and -CD8 mAbs rejected donor skin grafts by day 20, with a
median survival time (MST) of 10 days (Fig. 2A). All animals in the group
receiving pretreatment with all four mAbs had viable donor skin grafts
>72 days (p < 0.001 compared with graft
survival of mice that received only anti-CD4 and anti-CD8
mAbs). Mice that received pretreatment with anti-CD4, -CD8, and
-NK1.1 mAbs or anti-CD4, CD8, and Thy1.2 mAbs showed less prolonged
donor graft survival than the group receiving all four mAbs
(p < 0.05 and p < 0.01,
respectively), consistent with previous results (4). The
group that received pretreatment with anti-CD4, -CD8, and -NK1.1
had a MST of 26 days, whereas the group that received pretreatment with
anti-CD4, -CD8, and -Thy1.2 had little prolongation of donor skin
graft survival, with a MST of 10 days. These survival times were not
statistically different from that of the group receiving pretreatment
with only anti-CD4 and -CD8 mAbs. To evaluate immunocompetence in
these mixed chimeras and to determine whether the skin graft tolerance
shown was donor specific, the mice were grafted with nondonor
third-party SJL tail skin at the same time as donor skin. All groups in
the B6 wt mixed chimeras rejected third-party SJL skin grafts by day 16
(Fig. 2B). Overall, these results confirm our previous
observations in wt mice that in addition to anti-CD4 and -CD8 mAbs,
both anti-Thy1.2 and -NK1.1 mAbs are required to achieve long-term
chimerism and optimal tolerance (4).
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mouse chimerism and tolerance in B6
TCR-/- recipients
Fig. 3 shows that in
TCR-/- mice, pretreatment with anti-CD4
and -CD8 mAbs alone led to the lowest level of rat peripheral chimerism
at wk 3 post-BMT (mean 6.0%, range 0.4–19.9%). Mice pretreated with
anti-CD4, -CD8, -NK1.1, and Thy1.2 mAbs had the highest levels of
xenogeneic chimerism (mean 40.5%, range 32.5–45.6%) when compared
with the groups pretreated with anti-CD4 and -CD8 alone
(p < 0.001) or anti-CD4, -CD8, and -Thy1.2
(p < 0.01). Mice pretreated with anti-CD4,
-CD8, and -Thy1.2 mAbs had very low levels of rat peripheral chimerism,
which were similar to those in the group that received only
anti-CD4 and -CD8 mAbs (mean 7.8%, range 1.5–25.4%). The group
of mice pretreated with anti-CD4, -CD8, and -NK1.1 mAbs had a level
of xenogeneic PBL chimerism (mean 40.2%, range 15.9–56.0%,
p < 0.05) that was much higher than that in the group
receiving anti-CD4 and -CD8 mAbs alone, and that was similar to the
levels observed in the group receiving all four mAbs. Similar results
were obtained for gated rat monocyte, granulocyte, and lymphocyte
lineages when analyzed separately (data not shown). Table I
shows the
levels of rat class I+ cell mixed chimerism in
groups of ratTCR-/- chimeras in three
separate experiments at 3 wk post-BMT. Similar patterns were observed
at 5 wk (Fig. 3).
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-/- recipients grafted with donor
skin at 9 wk post-BMT, the group pretreated with only anti-CD4 and
-CD8 mAbs all rejected their F344 skin grafts by day 10, with a MST of
8 days (Fig. 4A). The longest
prolongation of donor skin graft acceptance was seen in the groups
receiving pretreatment with all four mAbs and in those receiving
anti-CD4, -CD8, and -NK1.1 mAbs. Both of these groups had a MST
>100 days, consistent with the peripheral chimerism results (Fig. 3).
These survival times were statistically different from those of the
group receiving pretreatment with only anti-CD4 and -CD8 mAbs
(p < 0.01). The group pretreated with
anti-CD4, -CD8, and -Thy1.2 mAbs had slightly less prolongation of
donor skin graft acceptance (MST = 74.5 days) than the groups
receiving anti-CD4, -CD8, and -NK1.1 mAbs, but graft survival was
longer than that in the group receiving only anti-CD4 and -CD8
mAbs. All groups of B6 TCR-/- recipients
showed immunocompetence by rejecting third-party A.SW grafts by day 14
(Fig. 4B). A repeat experiment showed similar results with
respect to donor skin, and the animals rapidly rejected third-party WF
rat skin grafts (data not shown). Thus, in
TCR-/- recipients, the administration of
anti-NK1.1 mAbs, in addition to anti-CD4 and -CD8
mAbs, is required and sufficient to achieve maximal long-term mixed
chimerism and tolerance. The administration of anti-Thy1.2 mAb had
little influence on mixed chimerism induction and had an intermediate
effect on donor-specific skin graft survival.
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mouse chimerism and tolerance in B6
TCR-/- recipients
Results in TCR-/- mice are
summarized in Fig. 5. At wk 3 post-BMT,
the group that had the highest level of rat peripheral chimerism was
the one that received pretreatment with all four mAbs (mean 18.4%,
range 12.6–28.7%). The group that was pretreated with anti-CD4
and CD8 mAbs alone had lower levels of rat peripheral chimerism at wk 3
(mean 8.6%, range 5.4–11.4%) than the group receiving all four mAbs
(p < 0.05). Both groups receiving pretreatment
with anti-CD4, -CD8, and -NK1.1 mAbs (mean 11.0%, range
9.5–13.0%) and anti-CD4, -CD8, and -Thy1.2 mAbs (mean 7.1%,
range 3.7–10.0%) had a level of rat peripheral chimerism that was not
significantly different from the group receiving anti-CD4 and -CD8
mAbs alone. A similar trend was seen at 5 (Fig. 5) and 7 wk (data not
shown) post-BMT. Similar results were obtained for gated rat monocyte,
granulocyte, and lymphocyte lineages when analyzed separately (data not
shown). Thus, in TCR--/-
recipients, the administration of both anti-NK1.1 and
anti-Thy1.2 mAbs is required to achieve long-term mixed chimerism.
Table I shows the levels of rat class I+ cell
mixed chimerism in groups of ratTCR-/-
chimeras in three separate experiments at 3 wk post-BMT.
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-/- mixed chimeras, all groups
of BMT recipients showed prolonged survival of donor F344 rat skin,
with a MST of 100 days (Fig. 6A). However, all groups of
BMT recipients rejected third-party WF rat skin grafts by day 26 (Fig. 6B). We evaluated these results further by grafting
nonreconstituted TCR-/- mice with rat skin,
and these mice accepted xenogeneic F344 rat skin and WF rat skin beyond
day 100 (data not shown). These results suggest that the third-party WF
rat skin graft rejection seen in the B6
TCR-/- chimeras is mediated by donor F344 T
cells circulating in the periphery of these animals (Fig. 7). Consistent with this observation, all
mice had rat T cells in the peripheral blood (Table II). These cells were tolerant to self
(rat F344) (Fig. 6A) and to the recipient mice (no evidence
of graft-versus-host disease (GVHD) was seen in any group) but not to
WF rat, and could therefore reject third-party nondonor WF rat skin
grafts (Fig. 6B). Table II summarizes the levels of rat T
cell mixed chimerism in four groups of ratwt,
ratTCR-/-, and
ratTCR-/- chimeras at 3 and 5 wk
post-BMT.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells and T cells, which express the
Thy1.2 molecule. Also, the xenoresistant cell population might be NK
cells, which express the NK1.1 molecule and, when activated, also
express Thy1.2, or T/NK cells, which express both NK1.1 and Thy1.2
molecules (13, 14, 15, 17, 18, 19, 20, 21, 30). Our study provides strong
evidence that T cells and NK cells are the major
CD4-CD8- xenoresistant
cell populations targeted by anti-Thy1.2 and anti-NK1.1
mAbs.
We have previously demonstrated that the depletion of host NK cells
before and following administration of allogeneic marrow led to only
slightly enhanced donor allogeneic pluripotent hemopoietic stem cell
engraftment, indicating that NK cells do not pose a major barrier to
allogeneic pluripotent hemopoietic stem cell engraftment when a
conventional marrow dose is given (31). However, the
depletion of NK1.1+ cells (NK cells) is necessary
in mice to achieve high levels of xenogeneic rat PBL chimerism,
indicating that NK cells play a major role in resisting rat marrow
engraftment. In both of the B6 TCR-/- and B6
TCR-/- groups pretreated with anti-CD4,
-CD8, and -Thy1.2 mAbs, very low levels of donor rat peripheral
chimerism were observed. In addition, there was only intermediate
prolongation of donor-type skin graft acceptance in the B6
TCR-/- group. In these treated animals, NK
cells were not depleted, demonstrating the powerful ability of NK cells
to reject rat hemopoietic cells. This is especially clear in the B6
TCR-/- mice, which do not have T/NK
cells, leaving NK cells as the only cell population depleted by
anti-NK1.1. Because the addition of this Ab increased the chimerism
and skin graft acceptance in TCR-/- mice to levels
seen in recipients of all four mAbs, NK cells clearly play an important
role in resisting rat marrow engraftment in mice. We speculate that the
more powerful ability of murine NK cells to reject rat marrow than
allogeneic marrow is due to a higher level of cross-reactivity of NK
cell inhibitory receptors on allogeneic than xenogeneic MHC Ags. The
importance of this cross-reactivity in reducing NK cell-mediated
resistance to allogeneic marrow grafts is underscored by the much
greater resistance to
2-microglobulin-deficient marrow than to
allogeneic marrow observed in lethally irradiated mice (32, 33). To our knowledge, cross-reactivity of murine NK cell
inhibitory receptors on xenogeneic MHC molecules has not been reported,
whereas recognition of rat MHC by the murine activating receptor Ly-49D
has been reported (34). In addition, xenogeneic
cross-reactivity of human NK cell inhibitory receptors on porcine MHC
ligands has been shown to be absent when examined
(35, 36, 37, 38). These observations suggest that engineering a
xenogeneic donor to express human MHC molecules or marked depletion of
host NK cells may be critical to the success of BMT as an approach to
xenogeneic tolerance induction.
Although anti-Thy1.2 mAb increased chimerism above that in
recipients of anti-CD4 and -CD8 alone in B6 wt animals, the
addition of anti-Thy1.2 mAb did not significantly increase
peripheral chimerism in B6 TCR-/- mice
treated with anti-CD4 and -CD8 mAbs. These results demonstrate an
important role for T cells in resisting donor rat marrow in wt
mice. In the B6 TCR-/- group pretreated with
anti-CD4, -CD8, and -NK1.1 mAbs, the only nondepleted
Thy1.2+ cell population would be T cells.
The low rat cell peripheral chimerism in this group provides further
evidence that mouse T cells play an important role in inhibiting
rat BMC engraftment. This, to our knowledge, is the first direct
demonstration that uncultured cells play a biological role in
the resistance to bone marrow engraftment or to xenografts.
Although our results demonstrate a role for T cells in
resistance to xenogeneic marrow grafts, they do not identify the
mechanism of inhibition. The mechanism could potentially be a direct
lytic effect of cells on donor BMCs. Murine T cells have
been implicated in rejecting allogeneic skin grafts that are disparate
in Qa-1 nonclassical MHC Ags (39), and in vitro activated
murine T cells have been shown to be cytolytic against rat
target cells (40). Furthermore, it has been demonstrated
that large numbers of cultured donor T cells could promote
engraftment and enhance hemopoietic reconstitution in allogeneic marrow
transplant recipients (41) and that transgenic T
cells could cause GVHD by specific recognition of nonclassical class Ib
Ags in mice (42). T cells might mediate rejection
not only through cytolysis but also via regulation of the immune
responses of other cells. T cells can secrete cytokines that fit
both a Th1 and Th2 profile (43, 44, 45), and can regulate
IFN- and TNF- production by NK cells and macrophages,
respectively (46, 47, 48).
Although NK1.1- Thy1.2+
cells resisting rat marrow engraftment in
TCR-/- mice were presumably T cells,
the xenoresistant cell population could also include
CD4-CD8- T/NK
cells, as this cell population would be depleted in mice receiving
pretreatment with anti-CD4, -CD8, and -NK1.1 mAbs with or without
anti-Thy1.2 mAbs. Studies have shown that T/NK cells mediate
NK-like cytotoxicity and may contribute to acute GVHD in mice
(49).
The B6 TCR-/- mice receiving
pretreatment with anti-CD4, -CD8, and -Thy1.2 mAbs had very low
levels of rat peripheral chimerism that were not significantly higher
than those in the group receiving only anti-CD4 and -CD8
mAbs. However, the anti-CD4-, -CD8-, and -Thy1.2-pretreated animals
demonstrated intermediate prolongation of donor skin graft survival,
whereas the group that received pretreatment with only anti-CD4 and
-CD8 mAbs rapidly rejected donor-type skin grafts. The imperfect
correlation between long-term chimerism and transplantation tolerance
in this group of mice is as yet unexplained. Perhaps anti-Thy1.2
mAb depletes a population of NK1.1+ cells (e.g.,
NK/T cells or activated NK cells) that participate directly in
xenogeneic skin graft rejection. The ability of anti-NK1.1 mAb to
overcome xenoresistance suggests that double negative T cells
not expressing NK1.1 do not resist engraftment. Therefore, the relevant
cell population depleted by anti-Thy1.2 mAbs could be NK cells that
express Thy1.2. Certain subsets of NK cells, such as NK cell
precursors, and activated NK cells (lymphokine-activated killer cells)
express the Thy1.2 marker (30, 50). In addition, T/NK
cells that are
CD4-CD8-TCR+Thy1.2+NK1.1+
would also be depleted by this regimen. T/NK cells have been reported
to reject allogeneic bone marrow grafts (51, 52). However,
the approach used here does not allow us to definitely confirm a role
for T/NK cells in xenoresistance.
In the B6 TCR-/- chimeras, all groups
accepted donor-type skin, but rejected third-party WF skin grafts. This
result is surprising, as mice lacking TCR+
T cells reportedly accept allogeneic skin grafts (53).
Consistent with this observation, our studies in untreated
TCR-/- mice show that T cells are
required to reject rat skin grafts, as nonreconstituted B6
TCR-/- mice did not reject rat skin grafts.
Our data suggest that reconstitution with rat marrow permits the
maturation of rat CD4+ and
CD8+ T cells that are capable of rejecting
third-party rat skin. Indeed, in all B6
TCR-/- chimeras, we have detected rat (F344)
T cells. These cells appear to be specifically tolerant of the donor
and host, and confer immunocompetence to the recipient mice. These rat
T cells presumably developed in the host mouse thymus. Consistent with
this interpretation, we have previously demonstrated that
immunocompetence is quite high among T cells developing in xenogeneic
thymus grafts. Murine T cells developing in porcine thymus grafts are
phenotypically and functionally normal and are specifically tolerant of
donor-derived cells in vitro and in vivo (54, 55, 56). Normal
development of human T cells and donor-specific tolerance are also
observed for human T cells developing in transplanted porcine thymus
grafts in SCID mice (57). It is somewhat surprising that
there was no difference in the rejection time of third-party rat WF
skin grafts between rat to TCR-/- chimeras
and rat to wt B6 chimeras described previously (4). This probably
reflects the tolerance to shared xenodeterminants by recipient T cells
in wt mice, which leads to slightly prolonged third party skin graft
survival (4). It is possible that donor-derived rat T cells also confer
some of the immunocompetence observed in rat to wt B6 chimeras.
However, we expect that the recipient T cells (the majority of T cells
in wt mice) confer most of the immunocompetence in these animals. It is
possible that the higher level of rat class I+
chimerism observed in all four groups of both
TCR-/- and TCR-/-
recipients compared with wt recipients is a consequence of the
permanent immunodeficiency in these gene knockout animals, resulting in
more complete immunosuppression than that achieved with mAbs.
Our study provides clear evidence that NK cells and T cells
resist rat bone marrow engraftment in mice, and that double negative
T cells do not present a major barrier to rat hemopoietic cell
engraftment. Although other reports have suggested the involvement of
NK cells and T cells in the rejection of allogeneic cells, our
study is the first to demonstrate a role for T cells in
xenograft rejection and in bone marrow rejection. Therefore, T
cells join NK cells as an important innate immune element resisting
xenografts.
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Acknowledgments |
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Footnotes |
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2 B.N. and D.T.C. equally contributed to this manuscript.
3 Address correspondence and reprint requests to Dr. Megan Sykes, Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital, MGH East, Building 149-5102, 13th Street, Boston, MA 02129.
4 Abbreviations used in this paper: BMT, bone marrow transplantation; WBI, whole body irradiation; T cells, CD3+ cells expressing the TCR; TI, thymic irradiation; T/NK cells, T cells that express NK cell-associated markers; B6, C57BL/6; wt, wild type; F344, Fisher 344; WF, Wistar-Furth; TCD, T cell-depleted; WBC, white blood cell(s); FSC, forward angle scatter; SSC, 90° light scatter; MST, median survival time; GVHD, graft-versus-host disease; NK cells, CD3- NK1.1+ cells; BMC, bone marrow cells.
Received for publication July 31, 2000. Accepted for publication October 25, 2000.
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) had 100% skin graft survival (n =
4). All the animals that received only the anti-CD4 and -CD8 mAbs
(
) rejected their donor grafts by day 20 (n =
5). The groups that received anti-CD4, -CD8, and -NK1.1 mAbs (•)
all rejected their grafts by day 36 (n = 3). The
group receiving pretreatment with anti-CD4, -CD8, and -Thy1.2 mAbs
(
) had a MST of only 10 days (n = 4).
B, Survival of mouse skin allografts (SJL). All animals
rapidly rejected third-party SJL mouse skin allografts.
