Both Rejection and Tolerance of Allografts
Can Occur in the Absence of Secondary
Lymphoid Tissues
This information is current as
of July 31, 2017.
Cavit D. Kant, Yoshinobu Akiyama, Katsunori Tanaka,
Susan Shea, Yohei Yamada, Sarah E. Connolly, Jose
Marino, Georges Tocco and Gilles Benichou
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Copyright © 2015 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2015; 194:1364-1371; Prepublished online 22
December 2014;
doi: 10.4049/jimmunol.1401157
http://www.jimmunol.org/content/194/3/1364
The Journal of Immunology
Both Rejection and Tolerance of Allografts Can Occur in the
Absence of Secondary Lymphoid Tissues
Cavit D. Kant, Yoshinobu Akiyama, Katsunori Tanaka, Susan Shea, Yohei Yamada,
Sarah E. Connolly, Jose Marino, Georges Tocco, and Gilles Benichou
F
ollowing allotransplantation, the immune response is initiated by T lymphocytes activated in either a direct or an
indirect fashion (1). The direct alloresponse relies on the
recognition by recipient T cells of intact donor MHC molecules
displayed on donor APCs. This response is polyclonal, because it
involves up to 10% of the entire T cell repertoire, owing to the
high frequency of MHC determinants presented to T cells and the
presentation of a multitude of peptides by allogeneic MHC molecules (2). In contrast, host T cells activated via indirect allorecognition interact with processed donor-derived peptides presented
by self-MHC molecules on recipient APCs (3). The indirect alloresponse is oligoclonal in that it is mediated by a few T cell clones
recognizing dominant determinants on alloantigens (4, 5). Although
either of these pathways can lead to acute rejection of skin allografts (6), acute rejection of vascularized solid organ transplants is
essentially mediated through the direct pathway. Alternatively, indirect alloreactivity is considered the driving force behind chronic
allograft rejection (7–10), which is characterized by graft tissue
fibrosis and blood vessel obstruction (11, 12).
Cellular trafficking is an essential element of the process associated with alloimmunity and transplant rejection. Following
skin transplantation, donor dendritic cells emigrate via lymphatics
to the recipient draining lymph nodes (13) where they activate
naive recipient T cells, thereby initiating the direct alloresponse
(14–17). Alternatively, in the case of primarily vascularized
organs, such as hearts and kidneys, it is likely that donor APCs
Transplantation Research Center, Department of Surgery, Massachusetts General
Hospital and Harvard Medical School, Boston, MA 02114
Received for publication May 6, 2014. Accepted for publication November 23, 2014.
This work was supported by National Institutes of Health Grants R21AI100278 and
R03AI094235 (to G.B.).
Address correspondence and reprint requests to Dr. Gilles Benichou, Department of
Surgery, Massachusetts General Hospital, Thier 807, 55 Fruit Street, Boston, MA
02114. E-mail address: [email protected]
Abbreviations used in this article: aly/aly-spl2, splenectomized aly/aly; B6, C57BL/6;
CAV, cardiac allograft vasculopathy; MST, median survival time; TLO, tertiary lymphoid organ; TLS, tertiary lymphoid structure; TMEM, memory T cell; Treg, regulatory T cell; wt, wild-type.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401157
leaving the graft through the blood spread rapidly to various
lymphoid organs where they can activate T cells. In addition,
some studies suggest that vascularized allografts can be rapidly
infiltrated by some host pre-existing memory T cells (TMEMs)
(18). Indeed, unlike naive T cells whose homing is confined to
lymphoid organs, TMEMs traffic regularly through peripheral
tissues (19, 20). These TMEMs may be activated via direct recognition of MHC molecules on donor dendritic cells remaining in
the graft and presumably endothelial cells expressing MHC class
II and costimulatory molecules as a result of inflammation. This
process could account for the direct activation of some alloreactive T cells following organ transplantation (21, 22). In contrast, it is still unknown where and how donor Ags are acquired,
processed, and presented by recipient APCs to T cells for induction of the indirect alloresponse.
In this study, we investigated the role of secondary lymphoid
organs in the T cell–mediated alloimmune responses, rejection,
and tolerance of mice transplanted with fully allogeneic conventional skin grafts or primarily vascularized skin or cardiac transplants. We show that aly/aly mice devoid of lymph nodes and
Peyer’s patches develop direct, but not indirect, alloresponses and
acutely reject both skin and cardiac allografts. In contrast, splenectomized aly/aly (aly/aly-spl2) mice reject skin, but not cardiac,
allografts. Remarkably, aly/aly-spl2 mice having spontaneously
accepted heart transplants developed donor-specific tolerance
mediated by CD4+ T cells. Therefore, both transplant rejection
and tolerance can be induced in the absence of secondary lymphoid organs.
Materials and Methods
Mice and transplantations
Six- to eight-week-old aly/aly mice used in this study were autosomalrecessive mutants of C57BL/6 (B6) mice displaying a point mutation in
the gene encoding the NF-kB–inducing kinase (23). These mice lack lymph
nodes and Peyer’s patches. In turn, heterozygous aly/+ mice displayed
a normal immune system and secondary lymphoid organs. In some
experiments, we used aly/aly-spl2 mice that are considered devoid of all
secondary lymphoid organs. B6 (H-2b) mice, BALB/c (H-2d) mice, C3H
(H-2k) mice, and B6 MHC class II–knockout mice were purchased from
the Jackson Laboratory (Bar Harbor, ME). Mice were bred and maintained
at Massachusetts General Hospital animal facilities under specific patho-
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In this study, we showed that aly/aly mice, which are devoid of lymph nodes and Peyer’s patches, acutely rejected fully allogeneic
skin and heart grafts. They mounted potent inflammatory direct alloresponses but failed to develop indirect alloreactivity after
transplantation. Remarkably, skin allografts also were rejected acutely by splenectomized aly/aly (aly/aly-spl2) mice devoid of all
secondary lymphoid organs. In these recipients, the rejection was mediated by alloreactive CD8+ T cells presumably primed in the
bone marrow. In contrast, cardiac transplants were not rejected by aly/aly-spl2 mice. Actually, aly/aly-spl2 mice that spontaneously accepted a heart allotransplant and displayed donor-specific tolerance also accepted skin grafts from the same, but not
a third-party, donor via a mechanism involving CD4+ regulatory T cells producing IL-10 cytokine. Therefore, direct priming of
alloreactive T cells, as well as rejection and regulatory tolerance of allogeneic transplants, can occur in recipient mice lacking
secondary lymphoid organs. The Journal of Immunology, 2015, 194: 1364–1371.
The Journal of Immunology
gen–free conditions. All animal care and handling were performed
according to institutional guidelines. “Classical” nonvascularized fullthickness skin allografts (2 3 3 cm) were placed on the groin area of
the recipients, according to the technique described by Billingham and
Medawar (24). Vascularized skin grafts were performed using a technique
described by our laboratory. Briefly, a 2 3 3-cm full-thickness flap was
outlined in the groin and elevated. The epigastric vessels were dissected,
the distal superficial and deep femoral vessels were ligated, and the femoral artery and vein were separated. The artery was flushed with 10%
heparinized saline until the venous flow in the pedicle became clear. Then
the femoral artery and vein were divided. For recipient mice, the samesized defect was created in the groin area. The femoral artery and vein,
right below the inguinal ligament, were separated and prepared for anastomosis. End-to-end anastomosis was performed for arteries, and end-toside anastomosis was performed for veins after the patency of the vessels
confirmed that the flap was sutured to the defect with interrupted sutures.
Vascularized heterotopic cardiac transplantation was performed as described by Corry et al. (25). Transplanted hearts were monitored daily by
palpation through the abdominal wall. Heart beat intensity was graded on
a scale of 0 (no palpable impulse) to 4 (strong impulse). Rejection was
defined by the loss of palpable cardiac contractions and verified by autopsy
and pathological examination.
T cell and T cell subset isolation
Preparation of sonicates
Stimulator spleen cells were suspended at 3 3 107 cells/ml in AIM-V
containing 0.5% FCS and sonicated with 10 pulses of 1 s each. The
resulting suspension was frozen in a dry ice/ethanol bath, thawed at room
temperature, and centrifuged at 300 3 g for 10 min to remove remaining
intact cells (1).
ELISPOT assays
Direct and indirect alloresponses by T cells were measured as previously
described (1). Briefly, 96-well ELISPOT plates (Polyfiltronics, Rockland,
MA) were coated with an anti-cytokine capture mAb in sterile PBS
overnight. On the day of the experiment, the plates were washed twice with
sterile PBS, blocked for 1.5 h with PBS containing 1% BSA, and washed
three times with sterile PBS. Responder cells or purified T cells were
added to wells previously filled with either intact donor cells (direct
response) or syngeneic APCs, together with donor sonicates (indirect
response), and cultured for 24 h at 37˚C, 5% CO2. After washing, biotinylated anti-lymphokine–detection Abs were added overnight. The
plates were developed using 800 ml AEC (Pierce, Rockford, IL; 10 mg
dissolved in 1 ml dimethyl formamide) mixed in 24 ml 0.1 M sodium
acetate (pH 5), plus 12 ml H2O2. The resulting spots were counted and
analyzed on a computer-assisted ELISA spot image analyzer (C.T.L.,
Cleveland, OH).
mAb treatments
For leukocyte-costimulation blockade, recipient mice were injected i.p. with
0.25 mg anti-CD40L mAbs (MR1) at the time of transplantation and at days
2, 4, and 6 posttransplantation, as previously described (26). For Treg
depletion, recipient mice were treated 1 d prior to transplantation with an
anti-CD25 Ab (PC61; 1 mg given i.p) that was shown to deplete CD25high
Foxp3+ Tregs in vivo (27). CD4+ or CD8+ T cells were depleted from recipient mice with anti-CD4 (GK1.5) or anti-CD8 (53.6.72) mAb (1 mg given
i.p. at days 23 and 21 pretransplant), respectively. NK cells were depleted
using the mAb NK1.1 (PK136; 0.6 mg) given i.p on day 22 pretransplantation. The depletions of CD4+, CD8+, and NK cells were .95%
(data not shown).
Histology
Cardiac transplants were fixed in 10% buffered formalin, embedded in
paraffin, coronally sectioned, and stained with H&E for evaluation of
cellular infiltrates and myocyte damage (acute rejection) by light microscopy. For assessment of chronic rejection, cardiac grafts were stained with
Verhoeff’s elastin (vessel arteriosclerosis scoring) or Mason’s trichrome
(evaluation of fibrosis). Arteriosclerosis was assessed by light microscopy,
and the percentages of luminal occlusion and intimal thickening were
determined using a scoring system, as previously described (28). Only
vessels that displayed a clear internal elastic lamina were included in the
morphometric analysis (five to seven vessels/section). All arteries were
scored by at least two examiners in a blinded fashion.
Statistics
All statistical analyses were performed using STATView software (Abacus
Concepts, Berkeley, CA). The p values were calculated using the paired t
test, and values , 0.05 were considered statistically significant.
Results
Rejection of allografts and alloresponses in the absence of
secondary lymphoid organs
First, we investigated the rejection of fully allogeneic BALB/c
(H-2d) skin and heart allografts in wild-type (wt) B6 mice (H-2b),
B6 aly/aly mice (lacking lymph nodes and Peyer’s patches), and
B6 aly/aly-spl2 mice. As shown in Fig. 1A and 1B, control wt B6
mice acutely rejected skin and cardiac transplants within 10–
12 d after placement. A slight prolongation of skin graft survival
was observed in aly/aly mice (median survival time [MST] = 16 d)
(Fig. 1A, 1B). In contrast, in aly/aly-spl2 mice, whereas skin
allografts also were acutely rejected (although in a delayed fashion, MST = 30 d), cardiac allografts survived indefinitely (Fig. 1A,
1B). Additionally, wt mice that had been splenectomized rejected
skin and heart allografts at the same pace as did control wt B6
mice (Fig. 1A, 1B). Of note, histological examination of accepted
hearts explanted from aly/aly-spl2 mice revealed pathological
features characteristics of mild chronic rejection detectable at day
50 posttransplantation (Fig. 1D). Therefore, rejection of cardiac
allografts requires the presence of either the recipient’s lymph
nodes or spleen, whereas skin allografts can be rejected in the
absence of any secondary lymphoid organ.
Unlike conventional skin grafts, cardiac transplants are vascularized
at the time of transplantation, a feature that contributes to their lower
immunogenicity and greater susceptibility to tolerance induction
(29). This prompted us to test whether primary vascularization of
skin grafts would prolong their survival in aly/aly-spl2 mice. As
shown in Fig. 1C, aly/aly-spl2 mice rejected vascularized skin
allografts at the same pace as conventional skin allografts (Fig. 1A).
Direct, but not indirect, alloresponses are induced in
transplanted aly/aly mice
Next, we measured T cell alloresponses in aly/aly mice transplanted with BALB/c skin allografts. Direct and indirect alloresponses by T cells collected from the recipient’s spleen were
evaluated 10 d after transplantation using a previously described
ELISPOT assay (1). As expected, transplanted wt B6 mice
mounted potent direct and indirect alloresponses that were mediated primarily by proinflammatory T cells secreting IL-2 and
IFN-g and a few T cells producing type 2 IL-4 cytokine but not
IL-10 (Fig. 2A). In contrast, in aly/aly mice, the direct alloresponse was dominated by T cells secreting IL-4 and IL-10 cytokines. In addition, aly/aly mice failed to mount an indirect
alloresponse (Fig. 2B).
CD8+ T cells reject skin allografts in aly/aly-spl2 mice
To identify the cells causing transplant rejection in recipients
devoid of secondary lymphoid organs, aly/aly-spl 2 mice were
injected with anti-CD4 (GK1.5)-depleting, anti-CD8 (56.8.72)depleting, or NK1.1 (anti-NK cells)–depleting Abs, as described in Materials and Methods. As shown in Fig. 3A, although depletion of CD4+ and NK cells had no effect, treatment
with anti-CD8 mAbs resulted in long-term survival of skin allografts. Therefore, CD8+ T cells mediate skin allograft rejection in
aly/aly-spl2 mice.
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T cells, as well as CD4+ and CD8+ T cell subsets, were isolated from the
spleen and lymph nodes of transplanted and naive mice by negative selection using commercially available T cell purification columns, according to the manufacturers’ instructions (Accurate Chemical & Scientific,
Westbury, NY; R&D Systems, Minneapolis, MN). Purified T cells were
washed in HBSS and used in ELISPOT assays.
1365
1366
ALLOGRAFT REJECTION WITHOUT LYMPHOID ORGANS
Detection of alloresponses in the blood and bone marrow of
transplanted aly/aly-spl2 mice
Because aly/aly-spl2 mice lacking all secondary lymphoid organs
reject skin allografts through a process involving CD8+ T cells, we
investigated whether an allospecific response could be detected in
other lymphoid tissues. To test this, T cells from the peripheral blood
and bone marrow of aly/aly-spl2 mice were tested 10 d after BALB/c
skin grafting for the presence of a direct alloresponse. As shown in
Fig. 3B, direct alloresponses were detected with T cells isolated from
bone marrow or peripheral blood. No response was detected in
nontransplanted aly/aly-spl2 mice (data not shown). These results
demonstrate that transplanted aly/aly-spl2 mice can mount an allospecific T cell response, despite the absence of lymph nodes and spleen.
To test this, aly/aly-spl2 mice that rejected a BALB/c skin graft
were retransplanted (20 d after rejection) with a skin allograft
derived from the same or a third-party C3H (H-2k) donor. First, we
observed an expansion of T cells expressing CD44 (mostly CD44+
CD62L2 effector TMEMs), which is characteristic of a memory
phenotype (Fig. 4A). Consistent with this observation, these mice
rejected a second BALB/c skin graft in an accelerated fashion
(Fig. 4B). No accelerated rejection was observed after placement
of control third-party C3H allografts (data not shown). Therefore,
despite their lack of secondary lymphoid organs, aly/aly-spl2
mice can mount a donor-specific memory alloimmune response
resulting in a second graft rejection.
aly/aly-spl2 mice mount memory alloresponses
Differential rejection of heart and skin grafts does not depend
on graft size
Next, we investigated whether aly/aly-spl2 mice can mount a donor-specific memory alloresponse following allotransplantation.
Minor Ag-mismatched (H-Y) heart transplants are less susceptible to rejection than are skin grafts due to their larger size,
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FIGURE 1. Allograft rejection in aly/aly and aly/aly-spl2 mice. wt B6, aly/aly, aly/aly splenectomized (aly/aly SPL-), and splenectomized B6 (B6 SPL-)
mice were transplanted with a BALB/c conventional skin allograft (A), a BALB/c heart transplant (B), or a primarily vascularized BALB/c skin allograft
(C). The results are shown as the percentage of graft survival over time after transplantation. Four to eight mice were tested in each group. Graft survival
was analyzed using the Kaplan–Meier method, and survival curves were compared using the log-rank test. (D) Representative photomicrograph (Tri
Masson blue, original magnification 340) of a BALB/c heart transplant harvested from an aly/aly mouse 50 d after its placement.
The Journal of Immunology
1367
CD4+ T cell–mediated tolerance to allografts in aly/aly-spl2
mice
Finally, aly/aly-spl2 mice, which had accepted a BALB/c heart for
50 d, were transplanted with a skin allograft from the same (BALB/c,
H-2d) or a third-party (C3H, H-2k) donor. Strikingly, BALB/c skin
allografts exhibited indefinite survival (.250 d), whereas C3H thirdparty allografts were rejected within 20–25 d posttransplantation
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FIGURE 2. Direct and indirect T cell alloresponses in transplanted
mice. The frequencies of T cells activated via direct (A) or indirect (B)
allorecognition and secreting proinflammatory cytokines type 1 (IL-2 and
IFN-g) or type 2 (IL-4 and IL-10) cytokines were measured by ELISPOT.
Alloresponses were measured using spleen T cells collected from naive B6
mice (no Tx) and from B6 and aly/aly mice that received a BALB/c skin
allograft (10 d posttransplantation). The results are expressed as mean
spots/million T cells (6 SD) for each cytokine. Five to eight mice were
tested in each group.
presumably causing T cell exhaustion (30). However, this
finding may only be relevant to transplants rejected via oligoclonal alloresponses, such as the anti–H-Y response. Nevertheless, given the overall paucity of lymphocytes in aly/aly-spl2
mice, we reasoned that the graft size could influence the rejection process. To address this question, aly/aly-spl2 mice
were transplanted with skin allografts of increasing sizes: 2
(size of control grafts), 4, and 9 cm2. Bigger-sized grafts were
rejected significantly faster than were the initial 2 cm2–sized
grafts (Fig. 4C). Therefore, larger skin grafts did not exhaust
T cells in aly/aly-spl2 mice, and they were also more immunogenic.
FIGURE 3. Mechanisms involved in alloskin graft rejection by aly/aly-spl2
mice. (A) aly/aly-spl2 mice were injected twice i.p. with anti-CD4 (GK1.5;
1 mg), anti-CD8 (53.6.72; 1 mg), or NK1.1 (PK136; 0.6 mg) mAbs at days
23 and 21 prior to placement of a BALB/c skin allograft. The results are
shown as the percentage graft survival over time after transplantation. Four
to eight mice were tested in each group. Graft survival was analyzed using
the Kaplan–Meier method, and survival curves were compared using the
log-rank test. (B) Direct alloresponses were measured using bone marrow
or peripheral blood T cells collected from aly/aly-spl2 mice that received
a BALB/c skin allograft (10 d posttransplantation). The frequencies of
T cells secreting proinflammatory type 1 (IL-2 and IFN-g) or type 2 (IL-4
and IL-10) cytokines were measured by ELISPOT. The results are
expressed as mean spots/million T cells (6 SD) for each cytokine. Three to
five mice were tested in each group.
1368
ALLOGRAFT REJECTION WITHOUT LYMPHOID ORGANS
(Fig. 5). Therefore, despite the absence of secondary lymphoid
organs, aly/aly-spl2 mice developed donor-specific tolerance. Of
note, heart transplants placed in aly/aly-spl2 mice displayed histological features of cardiac allograft vasculopathy (CAV) that
were detectable at day 50–75 posttransplantation (data not shown).
Therefore, ongoing chronic rejection of cardiac allografts did not
prevent tolerance of skin grafts.
Additional experiments were conducted to investigate the
mechanisms underlying transplant tolerance in aly/aly-spl2 mice.
First, BALB/c skin allografts were placed on aly/aly-spl2 mice
either at the time of (d0) or 14 or 30 d (d14 and d30) after
transplantation of BALB/c hearts. Skin grafts placed at day 0 or 14
were rejected acutely, whereas the majority of those transplanted
at day 30 exhibited long-term survival (Fig. 6A). Therefore, tolerance requires 2–4 wk to become established in this model. Next,
naive aly/aly-spl2 mice were injected with 106 CD4+ T cells isolated
from the peripheral blood of tolerant or control aly/aly-spl2 mice 3 d
prior to placement of a BALB/c skin graft. Adoptive transfer of
CD4+ T cells from tolerant mice resulted in long-term survival of
skin allografts in most recipients (Fig. 6B). Finally, aly/aly-spl2 mice
were injected with anti-CD4 Ab (GK1.5) or with anti-CD25 Ab
(PC61), using a regimen known to deplete CD4+CD25highFoxp3+
Tregs (27), 3 d prior to and 20 and 47 d after BALB/c heart transplantation. All mice were transplanted with a BALB/c skin graft 50 d
after cardiac transplantation. Recipients with anti-CD4 Abs accepted
heart, but not skin, transplants (n = 4, MST = 32 d). In contrast, antiCD25 mAb–treated mice accepted both transplants (n = 5, MST .
100 d). Altogether, these experiments show that, although transplant
tolerance is dependent upon CD4+ T cells, it apparently does not
depend on the presence of CD4+CD25highFoxp3+ Tregs.
Discussion
A single spontaneous autosomal mutation in the NIK gene on
chromosome 11 results in lack of lymph nodes and Peyer’s patches
in aly/aly mice (23). These mice display a disorganized thymic
architecture and severe defects in the spleen associated with the
absence of germinal centers, an atrophic white pulp, and no
marginal zone (31). These multiple defects result in alymphoplasia and compromised cell-mediated and humoral immunity (31,
32). Despite such severe immunodeficiency, it is remarkable that
aly/aly mice acutely rejected skin and cardiac allografts at the
same pace as did normal mice. Our observations are in contrast
with studies by Lakkis et al. (33) in the same aly/aly mouse
model; they reported similar rejection of heart transplants but
indefinite survival of skin allografts. Our results corroborate
reports from two other laboratories showing the rejection of skin
and lung allografts in aly/aly mice, as well as LTbR-knockout
mice, which also lack lymph nodes (34–37).
Direct, but not indirect, T cell alloresponses were detected in
skin-grafted aly/aly mice. This presumably reflects the fact that
T cell indirect allosensitization occurs through traditional priming
by peptides processed and presented by self-APCs in draining
lymph nodes (38). In contrast, direct allorecognition is unconventional in that it involves TCR interaction with intact allogeneic
MHC molecules present on foreign APCs and triggers a polyclonal response engaging up to 10% of the T cell repertoire (1, 39,
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FIGURE 4. Anamnestic T cell responses
and rejection of alloskin grafts of different
sizes by aly/aly-spl2 mice. (A) The frequencies of naive T cells (CD442CD62L+),
central TMEMs (TCM; CD44+CD62L+), and
effector TMEMs (TEM; CD442CD62L2)
were determined by FACS among lymphocytes isolated from the peripheral blood
of nontransplanted aly/aly-spl2 mice (top
panel) or from aly/aly-spl2 mice tested
2 wk (middle panel) and 4 wk (bottom panel)
after placement of a BALB/c skin allograft.
The results are representative of five mice
tested individually. (B) aly/aly-spl2 mice
were transplanted with a first skin graft at
day 0 and with a second graft at day 20 (after
rejection of the first graft). Ten mice were
tested. (C) aly/aly-spl2 mice were transplanted with BALB/c skin allografts of
different sizes (2, 4, or 9 cm2). In (B) and
(C), the results are shown as percentage
graft survival over time after transplantation. Graft survival was analyzed using the
Kaplan–Meier method. Three to eight mice
were tested in each group.
The Journal of Immunology
1369
these pre-existing TMEMs are unlikely to mediate the alloresponse based upon previous studies showing their inability
to trigger alloimmunity and allograft rejection upon adoptive
transfer in aly/aly mice (18). Finally, because high frequencies of
donor-specific activated inflammatory T cells were revealed in
the bone marrow of transplanted aly/aly-spl2 mice, we surmise
that this could represent the site of allosensitization. This hypothesis is supported by a recent study demonstrating that bone
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FIGURE 5. aly/aly-spl2 mice that spontaneously accepted a BALB/c
heart transplant developed donor-specific tolerance. aly/aly-spl2 mice that
spontaneously accepted a BALB/c heart for 50 d were transplanted with
a skin graft from the same (BALB/c) or a third-party (C3H) donor. The
results are shown as percentage skin graft survival over time after skin
transplantation (day 0). Graft survival was analyzed using the Kaplan–
Meier method. Three to five mice were tested in each group. The inset
shows a representative aly/aly-spl2 mouse that accepted a BALB/c skin
allograft for 280 d.
40). We surmise that, in skin-grafted aly/aly mice, donor APCs
leaving the graft upon its revascularization (days 4–5 posttransplant) can initiate a direct alloresponse given the exceptionally
high frequency of alloreactive T cells present in the recipient.
In contrast, indirect allosensitization requires “classical” T cell
priming in regional lymph nodes. This is supported by our recent report showing that primarily vascularized skin allografts,
whose dendritic cells emigrate through blood vessels, elicit direct,
but not indirect, alloresponses (29).
Unexpectedly, aly/aly-spl2 mice devoid of all secondary lymphoid organs acutely rejected skin allografts (Fig. 1). This process
was mediated primarily by CD8+ T cells that were activated
directly. Moreover, transplanted mice displayed a significant
generation/expansion of effector TMEMs and rejected a second
skin graft from the same donor in an accelerated fashion. Where
do T cells from transplanted aly/aly-spl2 mice encounter alloantigens? There is accumulating evidence suggesting that naive
alloreactive T cells can be primed either in secondary or tertiary
lymphoid organs (TLOs) located outside of the graft or in tertiary
lymphoid structures (TLSs) being formed within the graft itself
during inflammation (41–44). However, it is unlikely that naive
T cells of skin-grafted aly/aly-spl2 mice become sensitized in
TLOs or TLSs, because aly/aly-spl2 mice are devoid of TLOs
[with the exception of cryptopatches in the intestinal lamina
propria (42)] and never form TLSs in the skin, even upon lymphotoxin-a transgenesis (45). Another possibility is that the direct
alloresponse is mediated by “natural” alloreactive TMEMs already present in mice before transplantation and reactivated at the
graft site (18). Unlike naive T cells, TMEMs recirculate through
peripheral nonlymphoid tissues where they can be reactivated locally
upon Ag presentation (19, 20). Indeed, our study shows the presence
of 10–14% TMEMs in the blood of nontransplanted aly/aly-spl2
mice, presumably including some alloreactive cells. However,
FIGURE 6. Mechanisms involved in tolerance of skin allografts by aly/
aly-spl2 mice. (A) aly/aly-spl2 mice were transplanted with a BALB/c
heart and received a skin allograft from the same donor at the same time
(d0) or fourteen (d14) or thirty (d30) days later. The results are shown as
percentage skin graft survival over time after skin transplantation. Graft
survival was analyzed using the Kaplan–Meier method. Three to five mice
were tested in each group. (B) aly/aly-spl2 mice were injected with 106
CD4+ T cells isolated from the peripheral blood of tolerant (mice having
accepted a BALB/c heart for .100 d) or control naive aly/aly-spl2 mice
3 d prior to placement of a BALB/c skin graft. The results are shown as
percentage skin graft survival over time after skin transplantation (day 0).
Graft survival was analyzed using the Kaplan–Meier method. Three to six
mice were tested in each group.
1370
ALLOGRAFT REJECTION WITHOUT LYMPHOID ORGANS
marrow can serve as a priming site for T cell responses to bloodborne Ag (46).
Why do aly/aly-spl2 mice acutely reject allogeneic skin transplants but not heart transplants? Skin allografts are notoriously
more immunogenic and less susceptible to tolerogenesis than are
heart transplants (29). We showed recently that this is due, in part,
to the fact that cardiac allografts are immediately vascularized
after their placement, whereas conventional skin allografts are not
(29). However, our observation that primarily vascularized skin
allografts are acutely rejected in aly/aly mice (Fig. 1C) does not
support the view that graft vascularization accounts for the longer
survival of cardiac allografts in aly/aly-spl2 mice. Alternatively,
the presentation of some highly immunogenic skin-specific Ags
described by Steinmuller et al. (47) could account for the rejection
of skin, but not heart, allografts in aly/aly-spl2 mice.
Although aly/aly-spl2 mice did not acutely reject cardiac
allotransplants, histological examination of these grafts revealed
the presence of CAV typical of chronic rejection. No alloantibodies were detected in these mice (data not shown). It is conceivable that the few inflammatory T cells activated directly
(Fig. 3B, 200–300 IFN-g–producing cells/106 T cells) in these
mice were insufficient to ensure acute rejection of heart transplants but elicited CAV, a phenomenon previously documented
with adoptively transferred alloreactive T cell clones (30, 48). In
addition, we surmise that chronic rejection may be associated with
the high frequency of activated T cells secreting type 2 cytokines
(IL-4 and IL-10) that was detected in these mice. The contribution
of these T cells in chronic allograft rejection was documented in
several studies (49–51). Likewise, we recently reported that
donor-specific T cells secreting type 2 cytokines (IL-10) could
prevent early acute rejection of skin allografts while causing CAV
in an MHC class I–disparate mouse transplant model (8).
Remarkably, aly/aly-spl2 mice with cardiac transplants acquired donor-specific tolerance and accepted skin allografts from
the same, but not a third-party, donor. Two observations show the
contribution of CD4+ T cells to transplant tolerance in aly/aly-spl2
mice: pretreatment of recipient mice with anti-CD4 mAbs prevented tolerance induction (data not shown), and long-term survival of skin grafts was achieved in naive aly/aly-spl2 mice via
adoptive transfer of CD4+ T cells from tolerant mice (Fig. 6).
Therefore, in contrast to a previous study (33), graft acceptance in
this model does not result from immunologic ignorance but relies
on an active tolerance process. It is noteworthy that pretreatment
of aly/aly-spl2 recipients with an anti-CD25 mAb, which depletes
CD4+Foxp3+ regulatory T cells (Tregs), had no effect on transplant tolerance induction. This is not surprising, because it was
established that aly/aly mice are basically devoid of Foxp3+ Tregs
(52). In addition, studies by Bromberg and colleagues (53) support
the requirement of lymph nodes for Treg-mediated tolerance.
Therefore, it is unlikely that “conventional” Foxp31 Tregs are
responsible for tolerance to allografts in aly/aly-spl2 recipients. In
contrast, it is possible that Tr1 cells mediate tolerance. Indeed,
CD4+Foxp32 Tr1 cells secreting IL-10 are regularly activated
following systemic Ag administration via blood or oral routes (54–
57). Furthermore, donor-specific Foxp32 Tr1 cells were shown to
prevent allograft rejection (58–60). Therefore, we surmise that the
IL-10–producing alloreactive Tr1 cells present in tolerant aly/alyspl2 mice represent the cells that prevent acute rejection of skin
allografts by CD8+ T cells (8).
In summary, our study demonstrates beyond doubt that both
rejection and tolerance of allografts can occur in the absence of
secondary lymphoid organs. Both aly/aly and aly/aly-spl2 mice
mount direct, but not indirect, alloresponses. It is still unclear
where T cells actually interact with alloantigens on donor APCs
after skin transplantation of aly/aly-spl2 mice. We cannot rule out
the possibility that allorecognition by some pre-existing TMEMs
occurs in the graft. However, this hypothesis is not supported by
the alloresponse kinetics or by previous observations in the same
model. Alternatively, because we detected potent direct alloresponses in the bone marrow of transplanted aly/aly-spl2 mice, and
priming of naive T cells was documented previously in this tissue,
it represents a plausible site for initiation of the alloresponse in
transplanted mice lacking secondary lymphoid organs.
Disclosures
The authors have no financial conflicts of interest.
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