Download Transplant Outcome in Mice Effects of T Cell Frequency and Graft

Effects of T Cell Frequency and Graft Size on
Transplant Outcome in Mice
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J Immunol 2004; 172:240-247; ;
doi: 10.4049/jimmunol.172.1.240
http://www.jimmunol.org/content/172/1/240
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References
Chunshui He, Soren Schenk, Qiwei Zhang, Anna Valujskikh,
Jörg Bayer, Robert L. Fairchild and Peter S. Heeger
The Journal of Immunology
Effects of T Cell Frequency and Graft Size on Transplant
Outcome in Mice1
Chunshui He,* Soren Schenk,* Qiwei Zhang,* Anna Valujskikh,* Jörg Bayer,*
Robert L. Fairchild,*† and Peter S. Heeger2*†
I
mmune-mediated rejection of a transplanted organ involves
several discernable steps: activation of donor-reactive lymphocytes within secondary lymphoid organs (1), trafficking
of the activated T lymphocytes to the graft (2, 3), re-encounter
with the specific ligand at the graft site (4, 5), and elicitation of
effector functions that lead to target organ injury (4, 6 –9). If left
untreated, this induced antigraft T cell immune response generally
results in destruction of the transplanted organ. There are, however, selected situations in which transplantation does not result in
graft loss (10, 11) and in fact may lead to graft tolerance (12).
The specific factors that define whether antidonor T cell immunity is induced following transplantation and whether this induced
response will be pathogenic are not well understood. The number
and activation state of graft-derived dendritic cells capable of
priming recipient T cells (13, 14), the presence of ischemia-reperfusion injury to the graft (15, 16), and the number of foreign Ags
expressed by the graft (11, 17) are among the donor characteristics
that may contribute to the immunogenicity of a transplanted organ.
Once the recipient T cells are activated, potentially relevant factors
*Department of Immunology and Glickman Urologic Institute, Cleveland Clinic
Foundation, Cleveland, OH 44195; and †Institute of Pathology, Case Western Reserve
University, Cleveland, OH 44106
Received for publication August 13, 2003. Accepted for publication October
20, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a Scientist Development Grant from the American
Heart Association (to A.V.), an American Society of Transplantation Women’s and
Minority Faculty Grant (to A.V.), and National Institutes of Health Grant R01AI43578-02 (to P.S.H.).
2
Address correspondence and reprint requests to Dr. Peter S. Heeger, Department of
Immunology, Cleveland Clinic Foundation, NB30, 9500 Euclid Avenue, Cleveland,
OH 44195. E-mail address: [email protected]
3
Abbreviations used in this paper: NI, neointimal index; Treg, regulatory T cell.
Copyright © 2004 by The American Association of Immunologists, Inc.
that determine whether these lymphocytes will induce graft destruction include the quantity and distribution of Ag expressed on
the individual graft cells (4, 5, 17, 18), the subtype (CD4 vs CD8)
of T cells activated in response to the transplant (19), the specific
chemoattractant molecules that recruit T cells to graft site (2, 3),
the effector functions utilized by the activated T cells when they
re-encounter their ligand at the graft site (cytokines produced, cytotoxicity) (4, 8, 9, 17, 18, 20, 21), and the frequency of effector T
cells induced (11, 17, 22).
Transplantation of a fully allogeneic organ graft primes a high
frequency of donor-reactive CD4⫹ and CD8⫹ T cells capable of
mediating cytotoxic lymphocyte (CTL) activity and producing
type 1 proinflammatory cytokines (e.g., IFN-␥) (9, 17, 21, 23, 24).
Untreated recipients rapidly reject fully allogeneic heart and skin
grafts (10, 11, 22). In contrast, minor Ag-disparate transplants
prime lower frequencies of effector T cells, often with the same
proinflammatory phenotype as T cells induced to a fully allogeneic
graft (7, 10, 17, 22, 25, 26). Nonetheless, such minor Ag-disparate
grafts are rejected at a slower pace than MHC-disparate grafts, and
in some situations minor Ag-disparate transplants are not rejected
at all (7, 10, 17).
Although the frequency of induced effector T cells seems to
influence the outcome of a transplanted organ, it has been suggested that the size of the target organ might also contribute to the
resultant pathology (11, 27, 28). Proinflammatory, cytolytic, and
Th1 cytokine-producing effector T lymphocytes have limited life
spans and limited capacities to kill target cells. Thus, larger grafts
may be more resistant than smaller grafts to the consequences of a
defined number of activated donor-reactive effector T cells. Selected studies suggest that this may indeed be true (11, 27, 28), but
a detailed assessment of the interrelationship between T cell frequency and graft size has not been performed.
In an effort to better understand these relationships, we performed a series of experiments using minor Ag-disparate skin and
0022-1767/04/$02.00
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The features that determine whether graft-reactive T lymphocytes develop into effector cells capable of mediating organ destruction are not well understood. To investigate potential factors involved in this process, we first confirmed that female recipient mice
acutely rejected minor Ag-disparate male skin, but not heart transplants. Despite this difference in outcome, heart and skin
transplantation induced antidonor T cell responses of similar magnitude, specificity, and cytokine profile. The heart-graft-primed
T cells transiently infiltrated the graft and ultimately induced the development of chronic transplant vasculopathy. Increasing the
frequency of donor-reactive T cells by presensitization or by using TCR (CD8ⴙ antimale)-transgenic recipients did not mediate
acute rejection but accelerated the pace and severity of the vasculopathy. Surprisingly, decreasing the tissue mass of the donor
heart by 50% resulted in acute rejection of these smaller grafts without increasing the frequency of antidonor effector T cells in
the recipients. In complementary studies, placement of one or two male skin grafts on a single recipient did not affect the frequency
or cytokine profile of the induced antimale T cell repertoire. Nonetheless, the recipients of single grafts acutely rejected the
transplanted skin while the recipients of two skin grafts did not. These results provide new insight into the pathogenesis of
transplant vasculopathy and provide an explanation for the difference in outcome between murine skin and heart transplants by
highlighting the novel concept that the efficiency of transplant-reactive T cell immunity is heavily influenced by the tissue burden
it encounters at the effector stage. The Journal of Immunology, 2004, 172: 240 –247.
The Journal of Immunology
heart transplantation in mice. We found that skin and heart grafts
prime similar numbers of donor-reactive effector T cells and that a
threshold number of these effector T cells is required to result in
graft destruction. Consistent with previous work, larger numbers
of effector T cells are required to destroy a heart vs a skin graft
derived from the same donor strain. Our data additionally show,
however, that given a limited antidonor T cell repertoire, it is the
tissue mass of the graft rather than the type of graft (skin vs heart)
that determines whether the graft is accepted or rejected. Finally,
the data show that even in the absence of graft destruction, low
frequency antidonor T cell immunity has adverse effects on the
target organ and contributes to chronic graft injury.
Materials and Methods
Animals
Peptides
HYDbyp (NAGFNSNRANSSRSS), HYUtyp (NAGFNSNRANSSRSS),
and ␤gal96 –103 (DAPIYTNV) were synthesized by Research Genetics
(Huntsville, AL) at ⬎90% purity.
Placement and evaluation of skin and cardiac grafts
Full-thickness trunk skin grafts were placed using standard techniques (17,
23). Bandages were removed on day 7, and the grafts were then visually
scored daily for evidence of rejection. Grafts were considered fully rejected
when they were ⬎90% necrotic. Vascularized heterotopic cardiac grafts
obtained from 8- to 10-wk-old donors were placed in the abdomen as
described and palpated daily for evidence of a heartbeat (4, 8, 29). For
selected experiments, donor hearts were obtained from 3-wk-old donors.
Rejection was defined as a loss of palpable heartbeat. Technical failures
were defined as loss of heartbeat within 48 h of graft placement. Technical
failure rate was ⬍10% for standard heart grafts, but was ⬃40% in recipients of small, 3-wk-old donor hearts. Grafts were harvested at the time of
rejection or at predetermined time points posttransplant.
Histology and immunohistochemistry
Formalin-fixed paraffin sections of graft tissues were stained with H&E and
for elastin as described elsewhere (4, 8, 29). Vasculopathy was quantified
by calculating the neointimal index (NI) as described by Armstrong et al.
(30, 31) and by reporting the number of vessels with ⬎30% vasculopathy
per graft.
ELISPOT assays
Assays were performed as outlined previously in detail (4, 22, 29, 32).
Briefly, ELISPOT plates (Millipore, Bedford, MA) were coated overnight
with the capture Abs (obtained from BD PharMingen, San Diego, CA) in
sterile PBS, blocked with sterile 1% BSA in PBS, and washed three times
with sterile PBS. Spleen cells (0.2–1 ⫻ 106/well) were plated in HL-1
medium (BioWhittaker, Walkersville, MD) with or without mitomycin Ctreated male stimulator cells (400,000/well) and/or soluble Ags (HYDbyp,
HYUtyp, ␤gal96 –103 at 0.1–10 ␮M) and then incubated at 37°C in 5% CO2
for 24 h. After washing with PBS followed by PBS/0.025% Tween
(PBST), detection Abs (obtained from BD PharMingen) were added overnight. After washing with PBST, alkaline phosphatase-conjugated anti-biotin
Ab (Vector Laboratories, Burlingame, CA) diluted 1/2000 in PBST was added
for 90 min at room temperature. The plates were developed as previously
described (4, 22, 29, 32). The resulting spots were counted on an ImmunoSpot
Series 1 Analyzer (Cellular Technologies, Cleveland, OH).
In vivo CTL assays
This assay was adapted from published studies (33). Spleen cells were
isolated from naive B6 females and RBCs were removed by osmotic lysis.
The B6 spleen cells were suspended in RPMI 1640 plus 10% FBS at 10 ⫻
106/ml and pulsed with HYUtyp (experimental) or ␤gal96 –103 (control), 2
␮g/ml final concentration for 90 min at 37°C in 5% CO2. After washing
five times in PBS, cells were resuspended at 10 ⫻ 106/ml in PBS/0.1%
BSA. The control peptide-loaded cells were labeled with a high concentration of CFSE (20 ␮M) (CFSEhigh cells) and the HYUtyp-loaded target
population was labeled with a low concentration of CFSE (3 ␮M)
(CFSElow cells) for 10 min at 37°C in 5% CO2. The labeled cells were
washed three times in PBS. Equal numbers of cells from each population
were mixed together and injected i.v. into the tail vein of recipient mice at
predetermined time points posttransplantation. Injections were performed
such that each mouse received a total of 5 ⫻ 106 experimental (CFSElow)
cells and 5 ⫻ 106 control (CFSEhigh) cells in a total of 400 ␮l of PBS.
Sixteen hours later, the animals were sacrificed and the cell suspensions
were analyzed by flow cytometry. Each population was detected by its
differential CFSE fluorescence intensities. Up to a total of 500,000 gated
events were acquired per sample. For each animal, we calculated a ratio of
the number of CFSElow cells to the total number of CFSElow plus CFSEhigh
cells. Specific lysis was calculated as (1 ⫺ (ratio in naive B6 mice ⫺ ratio
experimental mice)) ⫻ 100.
Results
Consistent with previously published work (10, 11, 17), B6 female
recipients acutely rejected minor Ag-disparate male B6 skin grafts,
but male B6 heart grafts survived indefinitely in female B6 mice
(Fig. 1). Skin-derived Langerhans cells are more frequent in number and may be more immunogenic than heart-derived dendritic
cells (34, 35). In addition, because skin grafts are not initially
vascularized, induced ischemia-reperfusion injury is likely to be
more prolonged than in vascularized heart transplantation, possibly
leading to increased activation and immunogenicity of donor dendritic cells migrating to the recipient secondary lymphoid organs.
Potential consequences of these differences might be higher frequencies of anti-HY effector T cells and/or induction of more potent (pathogenic) T cell effector functions in recipients of skin vs
heart transplants. To assess this possibility, we analyzed the donorreactive T cell repertoire on day 14 posttransplant in skin vs heart
graft recipients. Fig. 2A shows donor-reactive IFN-␥ ELISPOT
recall responses for spleen cells obtained from individual skin or
heart graft recipients. Spleen cells from naive mice were included
as controls. We tested for reactivity to two known immune dominant determinants derived from the male Ag (36, 37), HYDbyp
(MHC class II, I-Ab-restricted) and HYUtyp (MHC class I, Dbrestricted), and for reactivity to mitomycin C-treated donor male
spleen cells. The frequency of donor-reactive CD4⫹ and CD8⫹ T
cells specific for male Ags was of similar magnitude in heart and
skin graft recipients. Although placement of a skin graft induced a
significantly higher frequency of donor-reactive IFN-␥-producing
T cells (⬃200 –250 per/million donor-reactive cells) than heart
graft recipients (⬃100 –150 per million), this difference was only
2-fold, could be partially attributed to differences in kinetics of
activation (see Fig. 3), and was of unclear biologic significance.
For comparison, the frequency of antidonor T cells induced in
response to fully allogeneic transplanted hearts or skin grafts (both
FIGURE 1. B6 females acutely reject syngeneic male skin but not heart
transplants. Graft survival is shown and median survival is statistically
different between groups by Kaplan-Meier analysis, p ⬍ 0.05.
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Male and female C57BL/6 (H-2b) and A/J (H-2a) mice, age 6 – 8 wk, were
purchased from The Jackson Laboratory (Bar Harbor, ME). Female
C57BL/10NA;-(Tg)TCR MataHari-(KO) Rag1 (H-2b, MataHari RAG KO)
(4), age 6 – 8 wk, were obtained as a generous gift from P. Matzinger
(National Institutes of Health, Bethesda, MD) and O. Lantz (Institut National de la Santé et de la Recherche Médicale, Paris, France). Female
MataHari RAG⫹/⫺ mice were obtained as the F1 generation of a cross
between MataHari RAG KO mice and wild-type B6 animals. All animals
were maintained and bred in the pathogen-free animal facility at the Cleveland Clinic Foundation.
241
242
FREQUENCY AND GRAFT SIZE AFFECT TRANSPLANT OUTCOME
acutely rejected) is 10- to 20-fold higher than to the minor Agdisparate grafts (Fig. 2A and Refs. 22 and 29). Immune cells from
skin or heart graft-primed mice did not produce IL-4 or IL-5 and
did not respond to control peptide ␤-gal96 –103 (data not shown). No
anti-HY immunity was detectable in naive B6 females (Fig. 2A).
To test whether another T cell effector function differed in mice
transplanted with skin vs heart grafts, we performed in vivo cytotoxicity assays. In this approach, the ability to mediate cytotoxicity
directly in vivo is determined by transferring specifically labeled
target cells along with a control target population into the transplanted or naive control recipients, and then the relative number of
specific target and control cells detectable in the spleens of the
FIGURE 3. Antimale immunity wanes with time. Antimale T cell immunity was tested by IFN-␥ ELISPOT (A) and in vivo cytotoxicity assay
(B) on days 7, 14, 21, and 30 posttransplant. The day 14 data are the same
data as shown in Fig. 2. Each point represents the mean response for three
to four animals per group tested at that time point. Error bars fell within the
size of the symbols for the cytotoxicity assay.
FIGURE 4. Chronic immune injury and transplant vasculopathy develop in male hearts transplanted into female recipients. Representative
H&E-stained sections from hearts obtained on days 7, 14, 21, 30, 60, and
90 posttransplant. Original magnification, ⫻10. Insets in the two lower
panels are elastin stains demonstrating endothelial cell proliferation consistent with transplant vasculopathy. Original magnification, ⫻10.
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FIGURE 2. Male heart and skin grafts prime similar antidonor T cell
immune responses. A, Frequencies of IFN-␥-producing cells reactive to
MHC class I-restricted HYUtyp (left), MHC class II-restricted HYDbyp
(middle), and male stimulator cells (right) per million splenocytes in heart
graft recipients (black circles), skin graft recipients (white circles), or naive
mice (gray circles). Each symbol represents the mean value of duplicate or
triplicate wells for an individual animal (⬍20% well-to-well variability).
Animals were sacrificed on day 14 posttransplant. Dashes are placed at the
mean value for each group. There was no response to control peptides
␤gal96 –103 or OVA323–339 (data not shown). The filled square represents the
mean value of donor-reactive IFN-␥-producing spleen cells tested at the
time of cessation of heartbeat (days 8 –10) in three B6 recipients of allogeneic A/J heart grafts. B, Representative flow cytometry plots for day 14
in vivo cytotoxicity assays are shown for a naive mouse (left), a skingrafted mouse (middle), and a heart-grafted mouse (right). Average percent
cytotoxicity for three to four animals per group is shown below each graph.
mice is determined by flow cytometry at a later time point. If
performed simultaneously in experimental and naive animals, one
can calculate the percent killing in vivo based on relative numbers
of detectable target cells in each. Syngeneic control target cells
loaded with ␤gal96 –103 or specific target cells loaded with HYUtyp
are labeled with different concentrations of CFSE so that they can
be differentiated by flow cytometry (a 5-fold concentration difference permits distinguishing between the two populations). For our
experiments, the HYUtyp-loaded targets cells were labeled with
CFSElow and the control syngeneic cells were labeled with CFSEhigh
. Equal numbers of the two populations (5 ⫻ 106 of each) were
then mixed and injected i.v. into the tail vein of a naive or a grafted
mouse. Animals were sacrificed the following day (⬍16 h) and
spleen cells were studied by flow cytometry for the presence of the
CFSE-labeled cells. As shown in Fig. 2B, skin and heart grafts
both induced potent antimale CTL activity compared with naive
controls. Thus, on day 14 posttransplant, the frequency, cytokine
profile, ability to mediate cytotoxicity, and relative immune dominance of the antimale T cell immune response did not differ substantially between skin and heart graft recipients. Nevertheless,
skin grafts were rapidly rejected but heart grafts were not (Fig. 1).
We next performed a kinetic analysis of T cell immune function
and graft histopathology following transplantation of male heart
grafts to syngeneic female recipients. As shown in Fig. 3, anti-HY
T cell immunity was readily detectable on days 7–14 posttransplant. The immune response to the MHC II-restricted peptide
peaked on day 7, several days before the detected response to the
dominant MHC class I-restricted peptide (as assessed by IFN-␥
ELISPOT (Fig. 3A) and in vivo CTL activity (Fig. 3B). Both antidonor CD4⫹ and CD8⫹ T cell immunity rapidly diminished by
days 21–30 posttransplant.
Kinetic histologic evaluation revealed a diffuse intragraft infiltrate detectable in male hearts placed into female recipients on
days 7–21 but few mononuclear cells were detectable in the recipients by day 30 (Fig. 4). Male heart grafts exhibited early vas-
The Journal of Immunology
243
Table I. Quantification of vasculopathy in recipients of B6 male heart grafts
Donor 3 Recipient
Male
Male
Male
Male
Male
Male
B6
B6
B6
B6
B6
B6
3 female B6 (n ⫽ 5)
3 female MataHari RAG⫹/⫺ (n ⫽ 5)
3 female B6 (n ⫽ 5)
skin followed by male B6 heart 3 female B6 (n ⫽ 3)
3 female B6 (n ⫽ 3)
3 male B6 (control, n ⫽ 3)
Average NI (%)
No. of Vessels with NI ⬎ 30
30 –35
30 –35
55– 63
50
87–94
88 –92
⬍10
30.5
22.9
42
38.2
13.7
0/26 (0%)
14/35 (40%)
8/43 (18.6%)
15/24 (62.5%)
14/33 (42%)
0/10 (0%)
primed antidonor effector T cells as assessed by IFN-␥ production
at the time of rejection (⬎11,000 per million spleen cells, n ⫽ 3,
data not shown). These results demonstrate that the male grafts do
express sufficient Ag to facilitate rejection by primed T cells if
large enough numbers of these effector T cells are present in the
recipients.
To further assess the role of T cell frequency as a mediator of
heart graft rejection, we next transplanted male B6 heart grafts into
MataHari RAG⫹/⫺ recipients. Female MataHari⫹/⫺ mice only
contain ⬃20% MataHari T cells (data not shown) with the remainder of the CD8⫹ and CD4⫹ T cell repertoire deriving endogenously. Despite the increased frequencies of donor-reactive T
cells compared with wild-type female B6 recipients (Table II), the
MataHari RAG⫹/⫺ recipients did not acutely reject male B6 heart
grafts. The grafts transplanted into these animals did, however exhibit clear evidence of chronic injury/vasculopathy by an early
30-day time point (Fig. 6 and Table I). ELISPOT recall assays
performed on days 7 and 30 posttransplant revealed 10- to 50-fold
higher absolute numbers of antidonor effector T cells compared
with wild-type B6 recipients (Table II). Overall, the ability of MataHari RAG KO females to reject male heart grafts is consistent
with the interpretation that a threshold number of T cells is required to acutely reject a cardiac transplant (11). The data further
suggest that subthreshold numbers of donor-reactive T cells mediate chronic injury and that the severity and rapidity of the induced injury is partially dependent on the frequency of donorreactive T cells in the recipient; below the threshold number that
FIGURE 5. Female recipients of male hearts are not tolerant to the male
Ag. Mean frequencies of splenic antimale IFN-␥ producers (A) and individual in vivo cytotoxicity assay results (B) are shown for female recipients
of male heart grafts 55– 60 days posttransplant. f and F, Results for recipients of male hearts alone (n ⫽ 4). 䡺 and E, Results for recipients of
male hearts that additionally received male skin grafts on day 30 following
the heart transplant. No responses were detected to control peptides (data
not shown). C, Representative H&E-stained and elastin-stained (inset) section of a male heart graft placed into a female recipient followed by placement of a male skin graft on day 30. The histology was assessed on day 55
after the heart transplant, 1 wk after the skin graft was visually rejected.
Inset is an elastin-stained section from the same graft. Original magnification, ⫻10.
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culopathy on day 60 posttransplant and progressive vasculopathy
by day 90. Quantitative analysis revealed significantly increased
frequency and severity of transplant vasculopathy in male hearts
transplanted into females when compared with isogenic female
heart grafts placed into female recipients by day 90 posttransplant
(Table I). The female grafts exhibited an essentially normal histologic appearance. The progressive vasculopathy in male but not
female grafts suggested that the female recipients of male heart
grafts were not tolerant to male transplantation Ags.
To formally assess the presence or absence of immune tolerance, male B6 heart transplants were placed into female B6 recipients and 30 days later, the animals were transplanted with male B6
trunk skin. The skin grafts were rejected by day 18 (n ⫽ 3, data not
shown), clearly showing that the female recipients were not tolerant to the male Ag. Recall assays performed 3 wk after skin graft
rejection (55– 60 days after heart graft placement) revealed antidonor immunity as assessed by both IFN-␥ ELISPOTs (frequency)
and in vivo CTL assays (Fig. 5), confirming the absence of tolerance. In striking comparison, there was minimal detectable antimale immunity in B6 female recipients of B6 male hearts grafts
alone on days 50 – 60 (no additional skin graft placed, Fig. 5).
Interestingly, the rejection of male skin did not precipitate acute
rejection of the previously placed male hearts; the heart grafts in
these animals continued to beat for more than 3 wk after placement
of the skin graft. Histology of these heart grafts performed on day
50, however, revealed more extensive vasculopathy compared
with male hearts placed into female recipients that did not receive
a skin graft (Fig. 5C and Table I). The mononuclear cell infiltrates
were more diffuse and the vasculopathy more severe in the animals
given male heart grafts followed by a subsequent male skin graft
compared with male grafts placed into females without a skin
graft. Overall, the data suggest that the heart transplant primed a
proinflammatory, antimale T cell immune response but that this
response was insufficient in quantity to mediate acute rejection of
the heart grafts. Nonetheless, the sublethal immune response led to
graft injury that eventually became manifest as chronic rejection.
“Boosting” of antimale T cell immunity via rejection of a male
skin graft was still insufficient to induce destruction of the heart
(over the 3-wk time period tested) but led to an accelerated form
of chronic graft injury.
An additional possibility that could account for the absence of
rapid graft destruction (persistent graft function) in female recipients of male hearts is that amount of male Ag expressed on the
parenchymal cells of the graft might be too low to be recognized
by primed T cells trafficking through the organ. To assess this
possibility, we placed male B6 heart grafts into female MataHari
RAG KO recipient mice. MataHari females bred onto a B6 RAG
KO background are TCR-transgenic animals in which every T cell
in the recipient is CD8⫹ and specific for the immune dominant
determinant HYUtyp plus Db (4). As shown in Fig. 6, MataHari
RAG KO females rapidly rejected the male (but not female) B6
heart grafts. As might be anticipated for a TCR-transgenic RAG
KO recipient, the rejection was associated with a high frequency of
Day Posttransplant
244
FREQUENCY AND GRAFT SIZE AFFECT TRANSPLANT OUTCOME
FIGURE 6. Increasing the frequency of antimale T
cells in the recipient accelerates the development of immune-mediated graft injury. A, Graft survival for male
hearts transplanted into female B6 MataHari RAG KO
recipients (‚) or B6 MataHari RAG⫹/⫺ recipients (䡺).
Graft survival was statistically different between the two
groups (p ⬍ 0.05). B, Representative H&E-stained sections (top) and elastin-stained sections (bottom) of male
B6 hearts harvested on day 30 after transplantation into
wild-type B6 females (left) or B6 MataHari RAG⫹/⫺ recipients (right). Original magnification, ⫻10 for H&E;
⫻20 for elastin-stained sections.
response was detectable in the lymph nodes draining the graft and
in the spleen. If two grafts were placed, the antidonor response was
detectable in both draining lymph nodes and the spleen, but the
overall number of antidonor T cells was the same as in the animals
with a single graft.
Discussion
It has long been noted that skin grafts are more readily rejected
than heart transplants by minor Ag-disparate recipients, but the
reasons for this difference in outcome has remained unclear (10,
11, 27, 38, 39). Skin grafts are thought to be more “immunogenic”
than cardiac allografts because they contain large numbers of
Langerhans cells (34, 35), a population of APCs with excellent Ag
presentation capability. Skin transplants may also induce a more
potent immune response than heart grafts because of the prolonged
ischemic injury they suffer while recipient vasculature grows in to
feed the graft (days for skin grafts vs hours for heart grafts). The
prolonged ischemia may provide inflammatory signals (16), including up-regulation of chemokines (40 – 44) that could increase
the efficiency of T cell priming and facilitate/accelerate trafficking
of the primed T cells to the graft.
We did find more antidonor T cells in the skin vs heart graft
recipients, consistent with the interpretation that skin is more immunogenic than heart tissue. Still, the highest detectable frequencies were much below the frequency of antidonor T cells induced
in response to a fully allogeneic graft (Fig. 2) and could in part be
related to differences in activation kinetics following skin vs heart
transplantation. More importantly, even a ⬎10- to 50-fold increase
in the frequency of donor-reactive effector T cells as found in the
MataHari RAG⫹/⫺ recipients (Fig. 6 and Table II) did not result in
acute rejection of male heart grafts. Thus, the detected difference in
the frequency of antidonor IFN-␥-producing T cells in skin vs
heart graft recipients was of unclear biologic significance and
alone could not account for the difference in graft outcome between the two sets of recipients. Also, there was no significant
difference in the relative immunodominance of MHC class I-restricted or MHC class II-restricted anti-HY responses between skin
Table II. Frequency and absolute number of anti-HYUtyp-specific IFN-␥ producers in recipients of male heart graftsa
B6 Male 3 MataHari B6 RAG⫹/⫺ Female
B6 Male 3 B6 Female
Day
posttransplant
Frequency (per 106
spleen cells)
Total per spleen
7
14
21
30
13 ⫾ 6
119 ⫾ 9
18 ⫾ 7
46 ⫾ 9
970 ⫾ 478
7,110 ⫾ 539
1,050 ⫾ 404
2,751 ⫾ 546
a
Day
posttransplant
Frequency (per 106
spleen cells)
7
2,955 ⫾ 525
187,740 ⫾ 41,940
30
1550 ⫾ 50
131,470 ⫾ 15,469
The results represent mean values ⫾ SE for four to six animals per group.
Total per spleen
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leads to acute organ destruction, higher frequencies of donor-reactive T cells induce chronic injury at a more rapid rate than lower
frequencies.
We next sought to directly determine whether the frequency of
donor-reactive T cells required to reject a heart graft was influenced by the size of the donor organ. We therefore challenged
wild-type female B6 mice with cardiac allografts from 3-wk-old
male donors. The weight of the hearts from these 3-wk-old donors
(52.2 ⫾ 11 mg) is 50% the weight of normal adult donors obtained
from mice 8 –10 wk of age (98 ⫾ 5.5 mg). Interestingly, ⬃60% of
the technically successful small heart transplants were acutely rejected by day 25 (Fig. 7). Immune-mediated injury was confirmed
by histologic examination in all grafts that stopped beating (Fig.
7). Recall immune responses on day 14 posttransplant revealed
that the frequency of antidonor T cells was no higher in recipients
of small vs large heart grafts (10 – 60 per million HYUtyp-specific
IFN-␥ producers and 30 –300 per million HYDbyp-specific IFN-␥
producers, n ⫽ 5, data not shown), showing that transplantation of
small vs large hearts was no more efficient at priming recipient T
cells. Thus, the data are consistent with the interpretation that the
ability of a given number of effector T cells to reject a target organ
is largely dependent on the size of the organ.
The above data suggest that the difference in outcome of skin vs
heart grafts (rejection vs acceptance) is the tissue burden of the
graft rather than the ability of a graft to prime recipient T cells. A
limited frequency of induced antidonor effector T cells seems to
only be able to destroy a graft of a given size (regardless of
whether it is a heart or a skin graft) before the response wanes. If
this is indeed true, then increasing the tissue burden of a skin graft
should prolong graft survival but should not affect the number of
primed effector antidonor T cells induced by the transplantation.
We assessed this possibility by placing either one or two 0.5-cm
male skin grafts onto wild-type recipient female mice. As shown in
Fig. 8A, skin graft survival was markedly prolonged in recipients
of two vs one skin graft. Moreover, placement of either one or two
skin grafts primed similar numbers of antidonor T cells in the
recipients. If one skin graft was placed, the antidonor immune
The Journal of Immunology
FIGURE 7. Female recipients reject syngeneic small male heart grafts.
A, Graft survival for male hearts obtained from 3-wk-old donors (‚) and 8to 10-wk-old donors (䡺) transplanted into B6 female recipients. Graft survival was statistically different between the two groups (p ⬍ 0.05). B,
Representative H&E-stained section of one small heart graft harvested on
the day of cessation of heartbeat. Original magnification, ⫻10.
FIGURE 8. Skin graft survival is affected by the graft tissue burden. A,
Survival of individual single male skin grafts (F) or double male skin
grafts (f) placed onto syngeneic female recipients is shown. Graft survival
was statistically different between the two groups (p ⬍ 0.05). B, The absolute number of IFN-␥-producing antimale spleen cells (calculated as frequency multiplied by the number of cells per lymphoid organ) are shown
in the recipients of either one or two skin grafts. Lymphoid organs from
three to four animals per group were pooled and the results depict the mean
values of duplicate or triplicate wells. There was no significant difference
in the total number of donor-reactive cells between groups. The experiment
was repeated with similar results.
These findings have important implications for human transplantation and suggest that understanding and monitoring the frequency of donor-reactive T cells may be relevant to predicting
long-term graft function in a clinical setting. In many respects, the
concept of antidonor T cell frequency as a risk for long-term outcome is reminiscent of the relationship between elevated blood
pressure and chronic organ injury (45, 46). Extremely high blood
pressure can result in acute injury (encephalopathy, renal failure,
heart failure) but subthreshold elevations in blood pressure produce chronic injury to the same organs. Moreover, the risk of
chronic injury is directly related to the level of blood pressure and
the time over which the blood pressure is elevated. Analogously,
our data, in conjunction with recently published work by our group
in human allograft recipients (47, 48), suggest that the frequency
of donor-reactive T cells in the recipient over time may directly
correlate with the risk of developing chronic injury.
One additional issue that we found particularly intriguing was
the observation that although anti-HY T cell immunity was induced by heart grafts, the immune response was not sustained in
wild-type recipient mice. Understanding in detail why such potent
immunity rapidly exhausts is an important issue that could potentially be exploited to prolong allograft survival in other situations.
One potential interpretation is that each effector T lymphocyte has
a limited life span and a limited ability to kill a certain number of
target cells. Once that limit is reached, the T cell dies. The number
of donor Ag-expressing cells in a heart graft (but not a skin graft)
may be sufficiently large to withstand the initial onslaught, with the
result being sublethal injury without graft destruction. New thymic
immigrants would only be primed inefficiently, as the number of
donor APCs capable of priming naive T cells is markedly reduced
over time. If an additional immunogenic stimulus such as a skin
graft is placed, then the new T cells (or memory T cells) expand,
reject the skin graft, and produce additional injury to the male
heart. Overall, such a model is analogous to the inadequate immune response induced to a chronic viral infection where the initial infection primes a response, but this antiviral immunity becomes “exhausted” over time (49, 50). Vaccination in the correct
context can boost the response and cure the infection.
An alternate but not mutually exclusive explanation for the observed decrease in antidonor immunity beyond 3 wk following
heart transplantation is that regulatory T cell responses develop as
a natural means of controlling the induced response. It has been
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and heart transplant recipients (Fig. 2). Nor was there a difference
in the development of type 1 cytokine secretion or the ability to
mediate CTL activity in recipients of heart vs skin grafts at 7–14
days posttransplant. Thus, our data strongly suggest that the immunogenicity of the donor graft alone cannot account for the difference in outcome between skin and heart grafts in this model.
The results delineated by this work raise the possibility that one
major difference between skin and heart grafts occurs not at the
priming stage, but instead at the effector stage. The larger amount
of tissue found in the heart vs the skin grafts seems to resist destruction by a limited T cell repertoire. This conclusion is supported by experiments showing that adequate Ag is expressed on
heart tissue to permit graft destruction if sufficient numbers of
high-affinity antidonor T cells are present in the recipient (Fig. 6).
Additionally, female recipients rejected small male heart grafts
despite the fact that they did not prime higher numbers of anti-HY
T cells than the larger grafts. Complementary experiments further
showed that increasing the amount of skin graft tissue resulted in
prolonged graft survival despite priming of the same frequency of
donor-reactive T cells as single graft recipients (Fig. 8). The results
confirm and extend previous studies in heart and skin graft models
suggesting that the size of the graft tissue can influence graft survival (11, 27, 28). Importantly, these previous studies did not assess whether the noted effect of tissue burden was attributable to T
cell priming or an influence at the effector stage issue, an issue
clarified by this work.
Our data also confirm, using a polyclonal system, the findings of
Jones et al. (11) in which the authors demonstrated that a threshold
number of TCR-transgenic T cells are required to reject an allograft and that this threshold number is larger for heart vs skin
grafts. The data from the present manuscript provide additional
insight by showing that a subthreshold number of donor-reactive T
cells, while not capable of acutely rejecting the graft, induce
chronic graft injury as manifested by fibrosis and transplant vasculopathy (Figs. 4 –7 and Table I). Male-to-female heart grafts
exhibited evidence of vasculopathy at 60 –90 days posttransplant,
while syngeneic female-to-female hearts were essentially normal
at this time point. If the frequency of antidonor effector T cells was
increased, but remained below the threshold required for rejection,
chronic injury occurred at an accelerated rate; vasculopathy was
detectable by day 30. This was true for both monoclonal TCRtransgenic T cells and for polyclonal effector T cells boosted by a
skin allograft. It is important to note that no anti-HY Abs develop
in this model and essentially no vasculopathy was detectable in
syngeneic female transplants. The resultant vasculopathy can
therefore only be attributed to antidonor (anti-HY) T cell
immunity.
245
246
FREQUENCY AND GRAFT SIZE AFFECT TRANSPLANT OUTCOME
Acknowledgments
We thank Earla Biekert and Alla Gomer for their superb technical
assistance.
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