Download prolongation of allograft survival in ccr7

0041-1337/04/7712-1809/0
TRANSPLANTATION
Copyright © 2004 by Lippincott Williams & Wilkins, Inc.
Vol. 77, 1809–1814, No. 12, June 27, 2004
Printed in U.S.A.
PROLONGATION OF ALLOGRAFT SURVIVAL IN
CCR7-DEFICIENT MICE
JAN H. BECKMANN,1 SHENG YAN,2 HEIKE LÜHRS,1 BETTINA HEID,1 SUSANNE SKUBICH,1
REINHOLD FÖRSTER,3 AND MATTHIAS W. HOFFMANN1,4
Background. Lymphocyte homing to secondary lymphoid organs is thought to be required for initiation of
the alloreactive immune response. Because CCR7 is
the essential chemokine receptor responsible for lymphocyte and dendritic cell homing to secondary lymphoid organs, allograft survival was analyzed in
CCR7-deficient (CCR7ⴚ/ⴚ) mice.
Methods. Heterotopic heart and skin allotransplantation was performed in CCR7ⴚ/ⴚ and wild-type (WT)
recipients. Graft survival was monitored daily. Grafts
and draining lymph nodes were analyzed by immunohistology and flow cytometry at different time points.
Groups of mice were splenectomized at the day of
allotransplantation.
Results. A significant though modest prolongation of
allograft survival in CCR7ⴚ/ⴚ recipients was observed
for heart grafts (WT, 7.3ⴞ0.5 days; CCR7ⴚ/ⴚ, 10.7ⴞ2.8
days) and skin grafts (WT, 8.9ⴞ0.9 days; CCR7ⴚ/ⴚ,
12.3ⴞ0.9 days). This was accompanied by a delay in the
cellular infiltration of allografts. T-cell accumulation
and expansion in the draining lymph nodes in
CCR7ⴚ/ⴚ recipients was severely impaired. Splenectomy had only a moderate prolongation effect on allograft survival in CCR7ⴚ/ⴚ mice.
Conclusions. These results suggest that CCR7-dependent processes support allograft rejection yet are
dispensable for the rejection response.
Initiation of organ allograft rejection is thought to occur in
secondary lymphoid organs (lymph nodes, spleen), where
alloreactive T lymphocytes and antigen-presenting cells convene (1). Migration of T cells and antigen-presenting cells
(APC) to secondary lymphoid organs is controlled by the
homeostatic chemokine receptor CCR7 and its ligands,
CCL19 (ELC) and CCL21 (SLC) (2). To prevent lymphocyte
homing and activation during allograft rejection, CCR7 thus
represents an ideal target. CCR7-deficient mice have been
generated by one of us, and show a severe defect in T-cell and
dendritic cell migration to secondary lymphoid organs, as
well as diminished delayed-type hypersensitivity responses
in vivo (3). They differ from mouse models, in which a different family of chemokine receptors, the inflammatory chemokine receptors, has been inactivated. Inflammatory chemokine receptors are involved in the effector phase of the
immune response. CXCR3-, CCR1-, CCR5-, and CXC3CR1deficient mice show a limited prolongation of allograft survival that is significantly augmented by the addition of the
immunosuppressive drug cyclosporine A (CsA) (4 –7).
The present study was performed to investigate the role of
T-cell and APC migration to secondary lymphoid organs during the initiation of the immune response. We demonstrate
that CCR7 deficiency extends skin and heart allograft survival but cannot ultimately prevent allograft rejection. Supporting the proposed mechanism of a defect in T-cell and APC
recirculation, activation of T cells in graft draining lymph
nodes was severely impaired in CCR7-deficient recipients. In
contrast to the effects observed in inflammatory chemokine
receptor-deficient mice, the immunosuppressive drug CsA
did not augment but rather abrogated the prolongation effect
of CCR7 deficiency on allograft survival.
MATERIALS AND METHODS
Mice
CCR7-deficient (CCR7⫺/⫺) and littermate control mice were originally on a mixed 129Sv(H-2b)/BALB/c (H-2d) background (3). 129Sv/
BALB/c mice were intercrossed, and H-2b homozygous offspring were
selected by flow cytometry. H-2b homozygous mice were used for
most experiments and are termed CCR7⫺/⫺ or littermate mice, if not
stated otherwise. For selected experiments, CCR7⫺/⫺ and littermate
control mice were backcrossed to C57/Bl6 (B6) for three generations
and are termed CCR7⫺/⫺ (B6) in the text. The latter mice had the
same H-2 (H-2b) as the original CCR7⫺/⫺ mice, but differed at minor
histocompatibility loci from the 129Sv/BALB/c mice. Therefore, these
mice were used to study the role of minor histocompatibility differences in heart- and skin-grafted mice. C3H/HeN (H-2k) mice served
as third-party controls. All mice were bred and maintained at the
Central Animal Facility, Hannover Medical School, under specificpathogen–free conditions. All experiments were approved by the
animal protection committee of the local authorities.
This work was supported by a grant from the Roche Organ Transplantation Research Foundation to M.W.H. and R.F., project grant
B10 of the SFB265 to M.W.H., and grant DFG Fo334/1-1 to R.F.
Operative Procedures
1
Department of Visceral and Transplantation Surgery, Hannover
Medical School, Hannover, Germany.
Heterotopic heart grafting was performed according to Corry et al.
2
Department of Visceral and Transplantation Surgery, Hannover (8). Pulmonary artery and aorta were anastomosed to the recipient’s
Medical School, Hannover, Germany. Currently, 1st Affiliated Hos- inferior vena cava and abdominal aorta, respectively. Graft function
pital, Zhejiang University, China.
was checked daily by palpation and was scored from 0 (no palpable
3
Institute of Immunology, Hannover Medical School, Hannover, heart beat) to 4 (strong, fast, rhythmic) according to Corry et al. (8).
Germany.
Cessation of heart function was confirmed by laparotomy.
4
Skin grafting was performed as described by Billingham and MeAddress correspondence to: Matthias W. Hoffmann, M.D.,
Ph.D., Visceral and Transplantation Surgery, Hannover Medical dawar (9). Recipients were anesthetized, shaved, and grafted with
School, 30625 Hannover, Germany. Email: hoffmann.matthias@ tail skin on the back or flank. Bandages were removed after 8 days,
mh-hannover.de.
and grafts were checked daily for signs of rejection. Grafts were
Received 21 December 2003.
scored as rejected when they had shrunk to less than a third of their
Revision requested 14 January 2004. Accepted 5 February 2004.
original size.
DOI: 10.1097/01.TP.0000131159.25845.EB
1809
1810
TRANSPLANTATION
Vol. 77, No. 12
Splenectomy was performed transabdominally during the heart
grafting procedure or through a left subcostal incision before skin
grafting. The splenic vessels were ligated with 8-0 silk, and the
abdominal wall and skin were closed in layers.
contralateral axillary and brachial lymph nodes. Draining lymph
nodes after syngeneic skin transplantation served as further
controls.
Immunosuppression
Mean survival time and standard deviation were calculated for
each group. Survival time between the two groups was compared by
the independent-samples t test (SPSS 9.0; SPSS, Inc., Chicago, IL).
P⬍0.05 was considered significant.
Statistical Analysis
Recipients were treated with subtherapeutic doses of cyclosporine
A (Sandimmune; Novartis Pharma, Basel, Switzerland). Four hours
after heart transplantation, mice were injected intraperitoneally
with 10 mg/kg of CsA in normal saline daily according to published
protocols (4, 5). Skin-grafted mice received daily doses of 20 mg/kg of
CsA administered intraperitoneally according to Larsen et al. (10).
RESULTS
Prolonged Survival of CCR7⫺/⫺ Skin and Cardiac
Allografts
Immunopathology
Skin and heart grafts were harvested at different time points,
embedded in Tissue Tek freezing medium (Reichert-Jung, Nuûloch,
Germany), snap-frozen, and stored in liquid nitrogen. Then, 5-␮m
cryostat sections (Frigocut 2800 E; Reichert-Jung) were air-dried
overnight and fixed in acetone at room temperature before hematoxylin-eosin staining (Merck, Germany; Sigma Chemical Co., St. Louis,
MO) or incubation with monoclonal antibodies. Primary and secondary antibodies were RM4-5/L3T4 (anti-CD4; PharMingen, San Jose,
CA), 53-6.7 (anti-CD8), RB6.8C5 (Gr-1; anti-Ly-6G, neutrophils), and
M1/70.15 (Mac1; anti-CD11b, macrophages). Antibodies were detected with horseradish peroxidase-conjugated goat anti-rat antibodies (Dianova, Hamburg, Germany), and were visualized with aminoethylcarbazole (Sigma, Deisenhofen, Germany). High-power fields of
200⫻ magnification were digitally scanned for counting immunostained cells of 20 fields (Olympus B202 microscope; Olympus Optical Co., Tokyo, Japan; AxioCam MRC; Carl Zeiss). Results are expressed as immunostained cells per field.
To determine the role of CCR7 in the rejection of primarily
vascularized heart and nonvascularized skin allografts, fully
major histocompatibility complex (MHC)-mismatched C3H
(H-2k) hearts and skin were grafted to CCR7⫺/⫺ mice and to
wild-type (WT) littermates (H-2b). Whereas WT mice rejected
C3H hearts within 7.3 days, allograft survival in CCR7⫺/⫺
mice was prolonged to 10.7 days (P⬍0.01) (Table 1). Similarly, rejection of C3H skin grafts was significantly faster in
WT (8.9 days) compared with CCR7⫺/⫺ recipients (12.3 days,
P⬍0.01) (Table 2). Similar results were observed in mice that
were matched at the MHC but mismatched at non-MHC loci.
To this end, CCR7⫺/⫺ and WT skin (H-2b, minor histocompatibility antigens derived from 129Sv and BALB/c) was
grafted to CCR7⫺/⫺ (B6) and WT (B6) mice (H-2b, minor
histocompatibility antigens derived from C57Bl/6). Whereas
WT recipients rejected minor histocompatibility disparate
skin grafts within 11.3 days, graft survival in CCR7⫺/⫺ mice
was significantly prolonged to 17.5 days (P⬍0.01) (Table 2).
To examine the role of graft-derived cells in allograft rejection (e.g., dendritic cells), CCR7⫺/⫺ hearts and skin (H-2b)
were grafted to MHC and minor disparate C3H recipients.
Neither heart-grafted (7.6 days in WT vs. 8.2 days in
CCR7⫺/⫺) nor skin-grafted mice (12.0 days in WT vs. 11.0
days in CCR7⫺/⫺) showed any signs of prolonged graft survival (Tables 1 and 2). Finally, deficiency of CCR7 in both
donor and recipient was analyzed in CCR7⫺/⫺ recipients by
transplanting allografts from CCR7⫺/⫺ mice. To this end
Flow Cytometry
At different time points after skin transplantation to the left flank,
recipients were killed. Left axillary and brachial (draining) lymph
nodes, right axillary and brachial (nondraining) lymph nodes, inguinal lymph nodes, and spleen were harvested, and isolated cells were
stained with the antibodies 53-6.7 (CD8FITC), GK1.5/L3T4 (CD4PE)
(Becton Dickinson, Franklin Lakes, NJ), KT3 (CD3FITC), and 1D3
(CD19bio). Streptavidin APC (Caltag Laboratories, Burlingame, CA)
was used as the secondary antibody to detect biotinylated primary
antibodies. Multiple color analysis was performed on a FACSCalibur
(Becton Dickinson). Absolute and relative numbers of immunostained cells per lymph node were compared between ipsilateral and
TABLE 1. CCR7–/– mice reject MHC-mismatched heart allografts in a prolonged fashiona
Donor
C3H (H-2k)
C3H (H-2k)
WT (H-2b)
CCR7–/– (H-2b)
Recipient
WT (H-2b)
CCR7–/– (H-2b)
C3H (H-2k)
C3H (H-2k)
Survival (days)
7,
7,
7,
5,
7,
8,
7,
7,
7,
9,
8,
8,
7,
9,
8,
8,
8, 8
10, 10, 12, 12, 14, 16
8
13
Mean⫾SD
p value
7.3⫾0.5
10.7⫾2.8
7.6⫾0.5
8.2⫾2.9
⬍0.01
NS
a
WT and CCR7–/– mice received C3H vascularized heart allografts.
NS, Not significant.
TABLE 2. Rejection of skin allografts in fully mismatched (donor, H-2k; recipient, H-2b) combinations and in non–MHCdisparate but MHC-matched combinations (donor, H-2b; minor H, 129Sv/BALB/c; recipient, H-2b; minor H, C57Bl/6)
Donor
C3H (H-2k)
C3H (H-2k)
WT (H-2b)
CCR7–/– (H-2b)
WT (H-2b)
WT (H-2b)
CCR7–/– (H-2b)
CCR7–/– (H-2b)
Recipient
WT (H-2b)
CCR7–/– (H-2b)
C3H (H-2k)
C3H (H-2k)
WT B6 (H-2b)
CCR7–/– B6 (H-2b)
WT B6 (H-2b)
CCR7–/– B6 (H-2b)
Survival (days)
8, 8, 8, 8, 8, 9, 9, 9, 10, 10, 10, 10
11, 12, 12, 12, 12, 12, 13, 13, 14
9, 11, 11, 13, 14, 14
10, 10, 10, 11, 12, 13
10, 10, 11, 11, 13, 13
14, 14, 14, 19, 21, 23
11, 12, 12, 12, 17, 17
19, 19, 21, 22, 23, 23
Mean⫾SD
p value
8.9⫾0.9
12.3⫾0.9
12.0⫾2.0
11.0⫾1.3
11.3⫾1.4
17.5⫾4.0
13.5⫾2.7
21.2⫾1.8
⬍0.01
NS
⬍0.01
⬍0.01
June 27, 2004
⫺/⫺
1811
BECKMANN ET AL.
b
CCR7
skin (H-2 , minor H: 129Sv/BALB/c) was grafted to
WT (B6) and CCR7⫺/⫺ mice (B6; H-2b, minor H: B6).
Whereas CCR7⫺/⫺ skin was rejected by WT recipients within
13.5 days, graft survival in CCR7⫺/⫺ recipients was significantly extended to 21.2 days (P⬍0.01) (Table 2). There was
no significant difference between the survival of WT and
CCR7⫺/⫺ skin grafts in WT recipients (11.3 days vs. 13.5
days, not significant [NS]) and between the survival of WT
and CCR7⫺/⫺ allografts in CCR7⫺/⫺ recipients (17.5 days vs.
21.2 days, NS) (Table 2).
Intragraft Leukocyte Infiltration in CCR7⫺/⫺ Mice
Heart allografts with a full MHC disparity (CCR7⫺/⫺ recipients and control littermates, C3H donors) were examined
by immunohistology at days 4 and 6 after transplantation. At
day 4, infiltration of CD4⫹ and CD8⫹ T cells was consider-
FIGURE 1. Analysis of heart graft infiltrating cells. (a) Reduced numbers of infiltrating CD8ⴙ cells after complete
MHC-mismatched heart transplantation to CCR7ⴚ/ⴚ compared with WT recipients (anti-CD8; magnification ⴛ200) (b)
Significant reduction of infiltrating CD4ⴙ and CD8ⴙ T cells at
day 4 after heterotopic heart transplantation in CCR7ⴚ/ⴚ
(open bars) vs. WT mice (closed bars). *P<0.05.
⫺/⫺
ably reduced in CCR7
recipients compared with WT controls. However, analyzing grafts 6 days after transplantation
revealed similar levels of leukocyte infiltration in both
groups (Fig. 1). Cell numbers of infiltrating neutrophils and
macrophages in CCR7⫺/⫺ recipients compared with WT mice
showed no significant differences at any time.
Lack of T-Cell Expansion in Draining Lymph Nodes of
CCR7⫺/⫺ Compared with WT Recipients after Fully
Mismatched Unilateral Skin Transplantation
To examine the intensity of the allogeneic immune response in CCR7⫺/⫺ mice, C3H skin was transplanted to the
recipient’s left flank. Recipients were killed at different time
points (days 4 and 7). Lymph nodes and spleen were harvested and analyzed by flow cytometry. As described previously (3), lymph node cell counts were reduced to a tenth in
CCR7⫺/⫺ mice compared with WT mice. The percentages of
CD4- and CD8-expressing cells were significantly lower in
CCR7⫺/⫺ mice (Fig. 2). After fully MHC-mismatched skin
transplantation (donor, C3H; recipient, CCR7⫺/⫺ or WT), cell
numbers in the draining lymph nodes of CCR7⫺/⫺ recipients
were 5 to 10 times lower compared with WT mice. Whereas
cell numbers in draining lymph nodes in WT recipient mice
increased at day 7, lymph node cells in CCR7⫺/⫺ mice showed
no further expansion. To exclude nonspecific effects of the
grafting procedure on the immune response, we grafted skin
to syngeneic recipients. On day 4 and day 7 after syngeneic
FIGURE 2. Leukocyte subsets in draining and nondraining
lymph nodes at different time points after unilateral MHCmismatched skin transplantation. Nondraining and draining
lymph nodes were analyzed 4 and 7 days after transplantation. Draining lymph nodes in CCR7ⴚ/ⴚ mice failed to expand
(right) compared with syngeneic (left) and allogeneic (center)
transplanted WT recipients.
1812
TRANSPLANTATION
Vol. 77, No. 12
grafting, increased cell numbers were observed in the draining lymph nodes. These were considerably lower than in
allografted mice but significantly higher compared with
CCR7⫺/⫺ mice (Fig. 2). In contrast, the number of lymphocytes in the spleen of recipients of syngeneic grafts showed no
significant differences in CCR7⫺/⫺ and WT 129Sv mice. As
shown previously (3), the number of CD4⫹ and CD8⫹ cells in
the spleen of CCR7⫺/⫺ mice was increased two- to threefold:
naive CCR7⫺/⫺ mice contained 23.2⫾11.3⫻106 CD4 T cells,
whereas syngeneically grafted mice contained 20.7⫾3.8⫻106
and allogeneically grafted mice 24.1⫾7.9⫻106 CD4 T cells. In
contrast, WT mice contained consistently fewer CD4 T cells
(naive, 10.0⫾2.5⫻106; syngeneic graft, 9.6⫾1.5⫻106; allogeneic graft, 6.8⫾1.8⫻106 CD4 T cells). A similar result was
observed for CD8⫹ cells in the spleen of CCR7⫺/⫺ recipients
(naive, 9.1⫾3.4⫻106; syngeneic graft, 8.9⫾0.1⫻106; allogeneic graft, 9.4⫾1.6⫻106 CD4 T cells) and WT recipients (naive, 5.3⫾0.3⫻106; syngeneic graft, 4.7⫾0.7⫻106; allogeneic
graft, 3.1⫾0.9⫻106 CD4 T cells).
Additional Prolongation of Allograft Survival in
Splenectomized CCR7⫺/⫺ Mice
The above results demonstrate that CD4 and CD8 T-cell
numbers are increased in the spleens of CCR7⫺/⫺ mice. In
accordance with previously reported results in alymphoid
aly/aly mice (11), which after splenectomy were unable to
reject allogeneic heart grafts, we attempted to extend allograft survival in CCR7⫺/⫺ mice by splenectomy. To this end,
WT and CCR7⫺/⫺ mice were splenectomized and grafted with
C3H skin. Compared with nonsplenectomized recipients,
splenectomy significantly prolonged the survival of skin
grafts in WT recipients by more than 4 days (8.9⫾0.9 days vs.
13.6⫾1.3 days, P⬍0.01) (Fig. 3). In CCR7⫺/⫺ mice, splenectomy extended skin graft survival by 5 days (12.3⫾0.9 days
vs. 17.3⫾2.2 days, P⬍0.01) (Fig. 3). Heart graft survival
could also slightly be enhanced by splenectomy in CCR7⫺/⫺
recipients (10.7⫾2.8 days vs. 13.3⫾1.0 days, P⬍0.05),
whereas WT recipients showed no significant increase in
graft survival (7.3⫾0.5 days vs. 8.3⫾1.2 days, NS) (Fig. 3).
Subtherapeutic Doses of CsA Abrogated the CCR7⫺/⫺ Effect
Synergistic effects between cyclosporine and the deficiency
of different chemokine receptors (e.g., CXCR3, CCR5,
CX3CR1, and CCR1) have previously been reported (4 –7). To
elucidate whether CsA might augment allograft survival in
CCR7⫺/⫺ mice, WT and CCR7⫺/⫺ mice, grafted with C3H
(H-2k) heart or skin, were treated with subtherapeutic daily
doses of CsA.
In WT mice, CsA did not significantly prolong heart allograft survival (7.8⫾1.8 days vs. 7.3⫾0.5 days without CsA,
NS). Interestingly, CsA abrogated the previously observed
prolongation of heart graft survival in CCR7⫺/⫺ mice
(8.0⫾2.0 days with CsA vs. 10.7⫾2.8 days without CsA, NS).
In contrast, in the presence of CsA, skin graft survival was
extended in both WT (15.5⫾2.4 days vs. 8.9⫾0.9 days without CsA, P⬍0.01) and CCR7⫺/⫺ mice (15.7⫾1.5 days vs.
12.3⫾0.9 days without CsA, P⬍0.05). Similar to the heartgrafted mice, there was no significant difference in allograft
survival between CsA-treated WT and CCR7⫺/⫺ mice
(15.5⫾2.4 days in WT vs. 15.7⫾1.5 days in CCR7⫺/⫺ mice,
NS) (Fig. 4).
FIGURE 3. MHC-mismatched (H-2k) allograft survival in WT
and CCR7ⴚ/ⴚ recipients (H-2b) with or without splenectomy.
(open diamonds) WT recipients; (filled diamonds) WT splenectomized recipients; (open squares) CCR7ⴚ/ⴚ recipients;
(filled squares) CCR7ⴚ/ⴚ splenectomized recipients. (a) Heart
graft survival time. (b) Skin graft survival time.
DISCUSSION
The above results show a significant though modest prolongation of allograft survival in mice lacking the chemokine
receptor CCR7. Because CCR7 is essential for T-lymphocyte
and APC migration to secondary lymphoid organs (3), this
limited effect indicates that homing to secondary lymphoid
organs accomplishes a nonessential role in allograft rejection. In this respect, the results resemble those obtained in
lymphotoxin-␣– deficient and lymphotoxin-␤-receptor– deficient mice that are devoid of lymph nodes and Peyer’s
patches. Splenectomized lymphotoxin-␣– deficient and lymphotoxin-␤-receptor– deficient mice reject fully allogeneic
heart allografts in a delayed fashion, but are not tolerant (12,
13). These results contrast to experiments by Lakkis et al.
(11), who used the alymphoid aly/aly mouse strain for similar
experiments. Splenectomized aly/aly mice were proposed to
June 27, 2004
BECKMANN ET AL.
FIGURE 4. MHC-mismatched (H-2k) allograft survival in WT
and CCR7ⴚ/ⴚ recipients (H-2b) with or without CsA treatment. (open diamonds) WT recipients; (filled diamonds) WT
recipients treated with CsA; (open squares) CCR7ⴚ/ⴚ recipients; (filled squares) CCR7ⴚ/ⴚ recipients treated with CsA. (a)
Mice that underwent heart transplantation received 10
mg/kg of CsA administered intraperitoneally per day. (b)
Skin-grafted recipients received 20 mg/kg of CsA per day.
represent a mouse model lacking secondary lymphoid organs,
and were tolerant to allogeneic skin and heart allografts.
Because both of these models, however, have severe defects
in other lymphoid compartments, it remained uncertain
whether the observed effects were mediated solely by the
absence of secondary lymphoid organs or might result from
support of hidden factors. The presented results obtained in
CCR7⫺/⫺ mice address the question of lymphocyte homing in
a model where lymph nodes and spleen are morphologically
intact but where deficiency of a single receptor, CCR7, impairs T-lymphocyte and APC migration into the secondary
lymphoid organs. Our results suggest that homing of these
cells by means of CCR7 is not an absolute requirement for
allograft rejection. Alternatively, it might be argued that
CCR7⫺/⫺ mice have developed CCR7-independent pathways
1813
of T-cell activation to fight environmental pathogens. This
view is supported by the finding that in lymph nodes and
spleen of CCR7⫺/⫺ mice a large proportion of T cells express
the activation and memory marker CD44 at high density (3).
Therefore, it appears possible that cross-reactive memory T
cells might be involved in the rejection of allografts in
CCR7⫺/⫺ mice.
It has been debated whether priming of alloreactive immune responses occurs in secondary lymphoid organs or in
the allograft itself. Results from this study document a limited effect of CCR7 deficiency on allograft survival, indicating
that homing to secondary lymphoid organs is not absolutely
required for rejection. However, it cannot be formally ruled
out that additional chemotactic factors or adhesion molecules
might compensate for the deficiency of CCR7. For instance,
L-selectin has been shown to be important for the adhesion of
leukocytes to high endothelial venules in secondary lymphoid
organs. Similar to our results in CCR7⫺/⫺ mice, L-selectin–
deficient mice rejected allogeneic skin grafts in a delayed
fashion (14). Furthermore, Tang et al. (14) reported a delay in
the cellular infiltration of skin allografts in L-selectin– deficient mice. Recent experiments by Kreisel et al. (15) support
the concept that nonhematopoietic cells within the allograft
can directly activate CD8⫹ T cells. Further on, they showed
that endothelial cells have the capacity to activate CD8⫹ T
cells in a B7-dependent fashion (16). These findings suggest
that nonprofessional APC, such as endothelial cells in case of
vascularized heart grafts, are sufficient to initiate the alloreactive immune response, independent of secondary lymphoid organs.
Dendritic cells (DC) have been shown to migrate from the
allograft to secondary lymphoid organs (17). The authors
speculated that DC migration from the allograft to SLO
might initiate the alloimmune response. Because CCR7 is a
key regulator of DC immigration into secondary lymphoid
organs, one aspect of the current study was to determine
whether CCR7-mediated migration of DC from the allograft
to SLO might affect the rejection response. Our results show
that CCR7⫺/⫺ skin and heart allografts were rejected with
similar kinetics as WT allografts. This indicates that CCR7mediated lymphocyte trafficking to SLO has no superior role
in allograft rejection. Further experiments aimed to determine whether CCR7 deficiency of both donor and recipient
might improve allograft survival. The results demonstrate
that in allografts differing in multiple minor antigens, allograft survival was not significantly prolonged in CCR7⫺/⫺
recipients of CCR7⫺/⫺ grafts compared with allograft survival of WT grafts in CCR7⫺/⫺ recipients. Therefore, CCR7mediated migration of passenger DC to secondary lymphoid
organs appears not to be essential to trigger an alloresponse.
Several chemokines and their receptors have been shown
to influence allograft survival. Distinct from the homeostatic
chemokine receptor CCR7, all of these are inflammatory
chemokines. The most pronounced effect was observed when
CXCR3 was inactivated in CXCR3-deficient mice or by antiCXCR3 monoclonal antibody therapy. MHC-disparate heart
allograft survival was prolonged to 58 days in CXCR3-deficient mice compared with 9 days in WT controls (4). Interestingly, permanent graft survival was observed when subtherapeutic doses of CsA were administered during the first
14 days after grafting. These results contrast with the findings in the present study. In CCR7⫺/⫺ recipients, the prolon-
1814
TRANSPLANTATION
gative effect of CCR7 deficiency in heart-grafted mice was
completely abrogated by the application of CsA. Similarly, in
skin-grafted mice, subtherapeutic CsA caused a significant
prolongation of skin graft survival in WT mice. In contrast,
addition of CsA in CCR7⫺/⫺ mice caused no prolongation
compared to untreated mice. These results suggest that CsA
had a negative rather than positive effect on allograft survival in the absence of CCR7. The reasons for this effect are
currently not clear. It may be speculated that CCR7-mediated allograft prolongation might involve an activity of responsive T cells, possibly with protective capacities, that
might be inhibited in the presence of CsA.
CONCLUSION
We have shown that CCR7 has a significant but overall
limited role in the prolongation of heart and skin allograft
survival in the mouse. This effect could be further enhanced
in splenectomized mice. Conventional immunosuppression
prevented rather than augmented the prolongation effect in
CCR7⫺/⫺ mice. The results suggest that recirculation of leukocytes to secondary lymphoid organs has a significant but
nonessential effect on the alloreactive immune response.
Acknowledgments. The authors thank M. Lipp for providing
CCR7⫺/⫺ mice, G. Bernhardt for critically reading the article, and C.
Ziegowski for excellent animal husbandry.
REFERENCES
1. Ascher NL, Hoffman RA, Hanto DW, et al. Cellular basis of allograft
rejection. Immunol Rev 1984; 77: 217–232.
2. Cyster JG. Chemokines and cell migration in secondary lymphoid organs.
Science 1999; 286: 2098 –2102.
3. Forster R, Schubel A, Breitfeld D, et al. CCR7 coordinates the primary
immune response by establishing functional microenvironments in sec-
Vol. 77, No. 12
ondary lymphoid organs. Cell 1999; 99: 23–33.
4. Hancock WW, Lu B, Gao W, et al. Requirement of the chemokine receptor
CXCR3 for acute allograft rejection. J Exp Med 2000; 192: 1515–1520.
5. Gao W, Topham PS, King JA, et al. Targeting of the chemokine receptor
CCR1 suppresses development of acute and chronic cardiac allograft
rejection. J Clin Invest 2000; 105: 35– 44.
6. Gao W, Faia KL, Csizmadia V, et al. Beneficial effects of targeting CCR5
in allograft recipients. Transplantation 2001; 72: 1199 –1205.
7. Robinson LA, Nataraj C, Thomas DW, et al. A role for fractalkine and its
receptor (CX3CR1) in cardiac allograft rejection. J Immunol 2000; 165:
6067– 6072.
8. Corry RJ, Winn HJ, Russell PS. Heart transplantation in congenic strains
of mice. Transplant Proc 1973; 5: 733–735.
9. Billingham RE, Medawar PB. The technique of free skin grafting in mammals. J Exp Biol 1951; 28: 385– 401.
10. Larsen CP, Elwood ET, Alexander DZ, et al. Long-term acceptance of skin
and cardiac allografts after blocking CD40 and CD28 pathways. Nature
1996; 381(6581): 434 – 438.
11. Lakkis FG, Arakelov A, Konieczny BT, et al. Immunologic ‘ignorance’ of
vascularized organ transplants in the absence of secondary lymphoid
tissue. Nat Med 2000; 6(6): 686 – 688.
12. Chin R, Zhou P, Alegre ML, et al. Confounding factors complicate conclusions in aly model. Nat Med 2001; 7(11): 1165–1166.
13. Zhou P, Hwang KW, Palucki D, et al. Secondary lymphoid organs are
important but not absolutely required for allograft responses. Am J
Transplant 2003; 3: 259 –266.
14. Tang ML, Hale LP, Steeber DA, et al. L-selectin is involved in lymphocyte
migration to sites of inflammation in the skin: Delayed rejection of
allografts in L-selectin-deficient mice. J Immunol 1997; 158: 5191–
5199.
15. Kreisel D, Krupnick AS, Gelman AE, et al. Non-hematopoietic allograft
cells directly activate CD8⫹ T cells and trigger acute rejection: An
alternative mechanism of allorecognition. Nat Med 2002; 8: 233–239.
16. Kreisel D, Krupnick AS, Balsara KR, et al. Mouse vascular endothelium
activates CD8⫹ T lymphocytes in a B7-dependent fashion. J Immunol
2002; 169: 6154 – 6161.
17. Larsen CP, Steinman RM, Witmer-Pack M, et al. Migration and maturation of Langerhans cells in skin transplants and explants. J Exp Med
1990; 172: 1483–1493.