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Pathologic Characteristics of Transplanted Kidney

Xenografts
Akira Shimizu,* Kazuhiko Yamada,* Simon C. Robson, David H. Sachs,*
and Robert B. Colvin
*Transplantation Biology Research Center and Department of Pathology, Massachusetts General Hospital/Harvard
Medical School, Boston, Massachusetts; Department of Pathology, Nippon Medical School, Tokyo, Japan; and

Department of Medicine, Transplant Center, Beth Israel Deaconess Medical Center/Harvard Medical School,
Boston, Massachusetts

ABSTRACT
For xenotransplantation to become a clinical reality, we need to better understand the mechanisms of
graft rejection or acceptance. We examined pathologic changes in a1,3-galactosyltransferase geneknockout pig kidneys transplanted into baboons that were treated with a protocol designed to induce
immunotolerance through thymic transplantation (n=4) or were treated with long-term immunosuppressants (n=3). Hyperacute rejection did not occur in a1,3-galactosyltransferase gene-knockout kidney xenografts. By 34 days, acute humoral rejection led to xenograft loss in all three xenografts in the long-term
immunosuppression group. The failing grafts exhibited thrombotic microangiopathic glomerulopathy
with multiple platelet-brin microthrombi, focal interstitial hemorrhage, and acute cellular xenograft rejection. Damaged glomeruli showed IgM, IgG, C4d, and C5b-9 deposition. They also demonstrated endothelial cell death, diffuse endothelial procoagulant activation with high expression of tissue factor and
vWF, and low expression of the ectonucleotidase CD39. In contrast, in the immunotolerance group, two of
four grafts had normal graft function and no pathologic ndings of acute or chronic rejection at 56 and 83
days. One of the remaining kidneys had mild but transient graft dysfunction with reversible, mild microangiopathic glomerulopathy, probably associated with preformed antibodies. The other kidney in the
immunotolerance group developed unstable graft function at 81 days and developed chronic xenograft
glomerulopathy. In summary, the success of pig-to-primate xenotransplantation may necessitate immune
tolerance to inhibit acute humoral and cellular xenograft rejection.
J Am Soc Nephrol 23: 225235, 2012. doi: 10.1681/ASN.2011040429

The use of animal organs might alleviate the critical

worldwide shortage of donor organs for clinical
transplantation.1,2 Miniature swine have been proposed as a suitable donor species because of their
anatomic and physiologic similarities to human
organs, breeding characteristics, and potential for
genetic modication.24 Transplantation of porcine organs into primates results in hyperacute rejection of the xenografts, with subsequent acute
humoral xenograft rejection (AHXR), also known
as acute vascular rejection or delayed xenograft rejection.16 Both hyperacute rejection and AHXR
are triggered by the binding of xenoreactive natural
antibodies to a specic epitope (galactose a1-3 galactose [Gal]) on the porcine vascular endothelium. 7,8 To date, considerable effort has been
J Am Soc Nephrol 23: 225235, 2012

applied to the depletion of anti-Gal antibodies

and inhibition of complement in pig-to-nonhuman
primate models.913 However, despite the successful prevention of hyperacute rejection by these
methods, with the return or continuing presence of
anti-Gal antibodies, all swine xenografts ultimately

Received April 29, 2011. Accepted August 29, 2011.

Published online ahead of print. Publication date available at
www.jasn.org.
Correspondence: Dr. Akira Shimizu, Transplantation Biology
Research Center, Massachusetts General Hospital, Building 1499019, 13th Street, Boston, MA 02129. Email: kaz.yamada@tbrc.
mgh.harvard.edu
Copyright 2012 by the American Society of Nephrology

ISSN : 1046-6673/2302-225

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succumb to AHXR, resulting in graft failure within a few

weeks.1013
To overcome this problem, a1,3-galactosyltransferase geneknockout (GalT-KO) pigs, which do not express the Gal epitope,
have been developed.1418 We have reported the clinical and immunologic outcomes of our initial trial of life-supporting kidney
xenotransplantation from GalT-KO pigs to baboons treated with
long-term immunosuppression (immunosuppression protocol)
or a protocol designed to induce tolerance though thymic transplantation (immunotolerance protocol).19 None of the grafts
succumbed to hyperacute rejection. However, all GalT-KO kidney grafts in animals receiving the immunosuppression protocol
failed within 34 postoperative days.19 In contrast, life-supporting
kidney grafts in the immunotolerance group survived 5683
days, with normal or only slightly elevated creatinine levels.19
The present study is an extension to the earlier work and was
designed to assess the pathologic characteristics of the GalTKO kidney xenografts in baboons treated with immunosuppression or immunotolerance protocols.

RESULTS
Clinical Findings in Animals

We have previously reported on the clinical outcomes of GalTKO kidneys in baboons treated with immunosuppression or an
experimental protocol designed to induce immunotolerance.19

In the present study, we examined the detailed pathologic features of kidney samples from the three baboons (B114, B126,
and B131) in the immunosuppression group and the four baboons (B113, B135, B118, and B134) in the immunotolerance
group; we have previously reported on all the animals.19 Hyperacute rejection did not develop in any of the GalT-KO grafts.
However, in the immunosuppressed baboons, graft dysfunction
developed at postoperative day 7 in all three grafts that showed
complete graft failure by postoperative day 34. In the immunotolerance group, the grafts were all prolonged but showed variable clinical courses. On the basis of clinical outcome, the animals
could be divided into three groups: One animal (B113) developed mild graft dysfunction from postoperative days 7 to 40,
with gradual improvement and persistent stable renal function
thereafter; a second animal (B118) showed mild and gradually
progressive graft dysfunction starting on postoperative day 50
and progressing to death on day 81; and two animals (B135 and
B134) showed persistently stable graft function beyond postoperative day 50. Baboon B134 survived with normal renal
function until postoperative day 83. All four immunotolerant
baboons died with functioning kidneys on postoperative days
56, 68, 81, and 83, respectively, from nonrenal causes (Table 1).
Acute Humoral Xenograft Rejection in
Immunosuppressed Baboons

In the early posttransplantation period at postoperative day 12,

IgM was clearly positive and IgG was weakly positive in glomeruli,

Table 1. Experimental groups, immunosuppression, graft survival, day of biopsies, and pathologic diagnosis
Animal ID

Protocols
(Thymus
Transplantation)a

Immunosuppressionb

Graftectomy
(Postoperative
Day)c

Death
(Postoperative
Day)

Cause
of Death

Morphologic
Changes

34

Arrhythmia
(K+ .10 mmol/L)

AHXR
(advanced)
AHXR
(advanced)
Glomerulopathy
(marked)

B114

IS

PTCD

33

B126

IS

TCD-2

B131

IS

PTCD

B113

TI (VTL)

TCD-1

68

Anesthetic problem

B135

TI (VTL)

TCD-2

56

Cardiac infarction

B118

TI (TK)

TCD-2

81

Pneumonia

B134

TI (TK)

TCD-2a

83

Heart failure

20

No obvious
rejection
No obvious
rejection
Mild chronic
graft injuries
No obvious
rejection

IS, immunosuppression; PTCD, partial T cell depletion regimen; AHXR, acute humoral xenograft rejection; TC2-2, T cell depletion regimen-2; TI, toleranceinducing; VTL, vascularized thymic lobe; TCD-1, T cell depletion regimen-1; TK, thymokidney.
a
Protocols: Life-supporting kidney transplantation was performed with long-term immunosuppression or tolerance-inducing protocols that were mediated by
thymic transplantation, vascularized thymic lobe, or thymokidney.
b
Immunosuppression: partial T cell depletion regimen: three doses of antithymocyte globulin plus three to four doses of rat antibody specic for human CD2
(LoCD2), anti-CD154 mAb, mycophenolate mofetil, and steroids; T cell depletion regimen-1 (thymectomy; splenectomy; and three doses of antithymocyte globulin
plus three to four doses of LoCD2, anti-CD154 antibody, mycophenolate mofetil, and steroids); T cell depletion regimen-2 (thymectomy, splenectomy, 100 cGy
whole-body irradiation on day 26, two doses of LoCD2, two doses of antithymocyte globulin, anti-CD154 antibody, mycophenolate mofetil, and steroids); and
T cell depletion regimen-2a, which was the same as T cell depletion regimen 2 but without steroids.
c
Graftectomy was performed when recipients demonstrated clinical evidence of graft rejection or severe deterioration of clinical condition, as evident by uremia
(BUN .100 mg/dl).

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with only minimal deposition noted in the peritubular capillaries (Figure 1). During the progression of graft failure by
postoperative day 34, deposition of IgM, IgG, C4d, and C5b-9
increased in glomeruli, small arteries, and peritubular capillaries.
On postoperative day 12, focal and segmental glomerulopathy
was noted with terminal deoxynucleotidyl transferasemediated
deoxyuridine triphosphate-biotin nick end-labeling (TUNEL)+
dead cells and CD41+ platelets, but interstitial and small arterial
injuries were minimal, with only a few TUNEL+ cells and CD41+
platelets (Figures 1 and 2). These ndings indicated that the
glomeruli were the rst site of anti-nonGal antibody deposition
and complement-mediated graft injury in AHXR in GalT-KO
kidneys. Progression of AHXR was characterized by diffuse,
global, and moderate-to-severe thrombotic microangiopathic

BASIC RESEARCH

glomerulopathy in all graftectomy samples by postoperative

day 34. The interstitium showed focal cellular inltration and
focal hemorrhage. TUNEL+ cells and CD41+ platelets were
prominent in the glomeruli, and a few were noted in the peritubular capillaries and arteries (Figures 1 and 2).
In the immunosuppression animals, thrombotic microangiopathic glomerulopathy was the characteristic pathologic
feature from the early post-transplantation period through the
progression of AHXR (Figures 24). Development of glomerulopathy was characterized by a rapid increase in IgM, IgG,
C4d, and C5b-9 deposition in glomeruli, along with multiple
thrombi formation, brin exudation, loss of capillaries with dead
and activated endothelial cells, mesangiolysis, and mesangial
proliferative lesions (Figures 24). The multiple thrombi seen
in injured glomeruli were CD41+ platelet-rich brin thrombi
present in glomerular capillaries, together with extensive deposition of IgM and C4d (Figure 3). The severity of glomerulopathy
and the extent of CD41+ platelet deposition correlated significantly with the deposition of C4d, indicating that thrombotic
microangiopathic glomerulopathy is a characteristic feature of
AHXR (Figure 2).
Lack of Acute Humoral Xenograft Rejection in
Immunotolerance Baboons

Figure 1. Pathologic features of the grafts during progression of

graft failure in the immunosuppression protocol. Immunouorescence studies of IgM (A and E: original magnication, 3200), IgG (B
and F: original magnication, 3200), C4d (C and G: original magnication, 3200), and C5b-9 (D and H: original magnication,
3200) in the grafts (B114) at postoperative days 12 (AD) and 33
(EH) showed progression of immunoglobulin and complement
deposition in glomeruli and small arteries and focally along peritubular capillaries. Development of graft failure, from postoperative
days 12 (I, K, and M) to 33 (J, L, NP), was associated with the
development of thrombotic microangiopathic glomerulopathy (I
and J: hematoxylin and eosin stain; original magnication, 3200)
with TUNEL+ dead cells (arrow) (K and L: TUNEL stain, original
magnication, 3200), CD41+ activated platelets (arrow) (M and N:
CD41 stain; original magnication, 3200), and brin exudation
(arrow) (O: Masson stain; original magnication, 3600). Interstitial
edema and focal hemorrhage (J) and brin thrombi (arrowhead) in
peritubular capillaries (P: Masson stain; original magnication, 3600)
were also evident in the interstitium at postoperative day 33.

J Am Soc Nephrol 23: 225235, 2012

In B113, an animal with initial mild graft dysfunction, the

deposition of IgM (but not IgG) was evident at postoperative day
13, together with the deposition of C4d and C5b-9 (Figure 5).
Mild microangiopathic glomerulopathy developed in the kidney. Cellular inltration was not prominent, and no obvious
alterations were seen in the tubulointerstitium or arteries. Although the deposition patterns of immunoglobulins and complement at postoperative day 29 were similar to those noted at
postoperative day 13, recovery of thrombotic microangiopathic
glomerulopathy was noted, together with repair of glomerular
capillaries in damaged glomeruli. In B118, an animal with late
unstable graft function, the deposition of IgM, C4d, and C5b-9,
but not IgG, was noted at postoperative 55, with mild microangiopathic glomerulopathy but no obvious cellular inltration
or arterial injury (Figures 4 and 5). At postoperative day 81 in
B118, focal chronic transplant glomerulopathy characterized by
focal segmental double contour of glomerular basement membrane and mesangial proliferative lesions developed (Figures 4
and 5), with minimal IgM, C4d, and C5b-9 deposition. Focal
interstitial brosis was noted, but arterial changes and acute
cellular xenograft rejection (ACXR) were not detected. The
presence of chronic transplant glomerulopathy and focal interstitial brosis indicated chronic graft injury with late unstable
graft function by postoperative day 81. In B134 and B135, animals that had stable graft function, no obvious thrombotic microangiopathic glomerulopathy developed; rather, the changes
were limited to focal IgM, C4d, and C5b-9 deposition in glomeruli (Figures 4 and 5). No IgG deposition was detected. Cellular rejection and arterial injury were not seen in the grafts.
These pathologic ndings demonstrated that AHXR, ACXR,
and chronic xenograft rejection did not develop in these grafts.
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proliferating cells, increased expression of

tissue factor and vWF, and decreased expression of CD39 on glomerular endothelial cells. In thrombotic microangiopathic
glomerulopathy, death of glomerular endothelial cells, loss of glomerular capillaries,
and activation with procoagulation status
of remaining endothelial cells developed
in the AHXR. In contrast, in the immunotolerance protocol, the glomeruli contained
no obvious TUNEL+ cells or PCNA+ cells,
no increased expression of tissue factor or
vWF, and intact CD31+ glomerular capillaries, together with CD39 expression on
endothelia cells.
Cellular Inltration in Grafts of
Immunosuppression and
Immunotolerance Baboons

ACXR developed in the grafts of the immunosuppression protocol (Figure 7).

During the early posttransplantation period, the cellular inltrate was composed
of polymorphonuclear leukocytes and
CD68+ macrophages, which might be associated with AHXR, in addition to a small
number of CD3+ T cells. In all grafts in the
immunosuppression protocol, the number
of inltrating cells increased after transplantation, and the inltrating cells comprised
CD3+ T cells and CD68+ macrophages, as
well as a small number of CD20+ B cells and
NK cells. Two-color immunohistochemisFigure 2. Pathologic differences between immunosuppression (B114, B126, and try identied the presence of TIA-1+ cytoB131) and immunotolerance (B113, B135, B118, and B134) protocols. The pathologic toxic granules in many inltrating CD3+ T
characteristics were evaluated semiquantitatively for the severity of glomerulopathy cells, indicating that they were cytotoxic T
(A); the degree of interstitial hemorrhage (B); the number of TUNEL+ dead cells in cells. Furthermore, CD3+ T cells inltrated
glomeruli (C) and peritubular capillaries (PTCs) (D); the glomerular deposition of IgM
the tubules, peritubular capillaries, and glo(E), IgG (F), C4d (G), and C5b-9 (H); the number of CD31+ glomerular capillaries (I); the
merular capillaries and were seen underdeposition of CD41 platelets (J); the expression of tissue factor (K), vWF (L), and CD39
neath the endothelial cells of small arteries,
(M); and the number of PCNA+ proliferating glomerular cells (N). Correlation between
C4d deposition in glomeruli and the severity of glomerulopathy (O) or CD41 deposition with ndings of tubulitis, capillaritis, acute
glomerulitis, and endothelialitis. In con(P) was also evaluated. POD, postoperative day.
trast, in the immunotolerance protocol,
only a few interstitial mononuclear cells
were detected and no or little tubulitis, capillaritis, glomeruliGlomerular Endothelial Cell Death and Activation,
tis, or endothelialitis was observed.
Associated with AHXR, in Immunosuppression and
Immunotolerance Protocols
We next examined glomerular cell death and endothelial Acute and Chronic Rejection Score in Banff
activation during the development of thrombotic microanClassication 2007
giopathic glomerulopathy in the immunosuppression and
In the xenografts (B114, B126, and B131) in the immunosupimmunotolerance protocols (Figures 2 and 6). The moderate
pression protocol, AHXR and ACXR developed by postoperto marked thrombotic microangiopathic glomerulopathy obative day 34, with a high acute rejection score according to Banff
served in the immunosuppression protocol was characterized
classication 2007 (Table 2).20 C4d deposition of peripheral
by the presence of many TUNEL+ cells, loss of CD31+ glomertubular cells was also evident because the C4d sore was 23.
ular capillaries, proliferating cell nuclear antigen (PCNA)+
On the other hand, in immunotolerance protocol, although
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C5b-9 deposits appeared in the grafts by

postoperative day 34, and the increase in
C4d deposition correlated strongly with
platelet-brin thrombi formation and the
development of thrombotic microangiopathic glomerulopathy. Focal interstitial
hemorrhage was also evident. These ndings indicate that AHXR develops even when
the donor is the GalT-KO swine. Although
the exact mechanism of AHXR is not yet
clear, it is likely that anti-nonGal antibodies
were involved.2124 In the present study,
anti-nonGal antibodies were primarily directed against endothelial cells because immunoglobulin was deposited in the capillary
walls. In this regard, several reports have recently suggested that the epitopes on pig
Figure 3. Development of thrombotic microangiopathic glomerulopathy and artegrafts recognized by human anti-nonGal
riolopathy in the immunosuppression protocol (A, F, and G: B114, postoperative day
antibodies include the swine leukocyte an33; B and H: B131, postoperative day 20; C and D: B126, postoperative day 34).
tigen, N-glycolyl neuraminic acid (NeuGc)
Thrombotic microangiopathic glomerulopathy developed with multiple thrombi
(arrowhead in A: periodic acid-methenamine [PAM] stain; original magnication, epitopes (Hanganatziu-Deicher antigen, al3600), brin exudation (arrow in B: PAM stain; original magnication, 3800), enlarged though this would not be a target in baboons
endothelial cells (arrow in C: PAS stain; original magnication, 3800) and mesangial because these animals also express this antiproliferative lesion (asterisk in D: PAM stain; original magnication, 3800). Almost all gen), and the Forssman antigen.25 Further
CD41+ platelet-rich thrombi were detected with brin; within vessels they were de- experiments are necessary to identify new
tected with deposition of IgM and C4d (double immunostaining against CD41 [red] target epitopes of these antibodies before
and green of brin [E], IgM [F] or C4d [G]; original magnication, 3600). Thrombosis consideration of strategies to prevent anti(arrow) was also evident in small arteries (H: PAM stain; original magnication, 3800). nonGal antibodymediated rejection, including the deletion or modication of
nonGal epitopes by genetic engineering of
the pig. Another possibility is suggested by several recent studone graft (B118) with unstable graft function had a mild chronic
ies demonstrating that Gal could not be completely eliminated
rejection score, the other three grafts with initial mild graft
by using current GalT-KO technology because the enzyme
dysfunction (B113) and stable graft function (B135 and B134)
iGb3 synthase can also synthesize Gal in GalT-KO mice and
had only a minimal acute and chronic rejection score.
pigs.21,26,27 However, because AHXR did not develop in the two
GalT-KO kidney grafts with stable graft function in the present
study, we consider it likely that their tissue expressed no Gal.
DISCUSSION
In thrombotic microangiopathic glomerulopathy, which is
similar to the glomerulopathy seen in human decay accelerWe examined the differences in the pathologic ndings of
ating factor-transgenic swinetobaboon kidney transplantaswine renal xenografts in long-term immunosuppression or
tion,28 both cell death and activation of endothelial cells were
immunotolerance protocols. We used GalT-KO Massachusetts
General Hospital miniature pigs that do not express the Gal noted and are probably related to immunoglobulin deposition
epitope as donors, thereby abrogating anti-Gal antibody
and complement activation. Cell death and activation of glomediated rejection. All three GalT-KO kidneys under the long- merular endothelial cells with the presence of TUNEL+ cells
term immunosuppression protocol were rejected by AHXR and and PCNA+ cells indicated that increased expression of vWF
ACXR by postoperative day 34; they were characterized pathand tissue factor, and decreased expression of CD39, may have
ologically by thrombotic microangiopathic glomerulopathy,
contributed to thrombotic formation because these changes are
focal interstitial hemorrhage, and cellular inltration. In conassociated with an inevitable shift from an anticoagulant to a
trast, two of four kidneys from the immunotolerance protocol procoagulant state, further compounded by intrinsic molecular
maintained normal graft function at postoperative days 56 and
barriers in thromboregulation.1 This type of glomerulopathy is
83, without pathologic ndings of AHXR, ACXR, or other
considered the next major hurdle for xenotransplantation.29
signs of chronic xenograft rejection.
The characterization of cellular rejection in vascularized
In our long-term immunosuppression protocol, AHXR and
xenograft models is still limited by the rapid development of
ACXR could not be overcome, despite intensive treatment.
AHXR. In our immunosuppression protocol, cellular inlDuring the progression of graft failure, IgM, IgG, C4d, and
tration developed in the xenografts. In addition to neutrophil
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Figure 4. Electron microscopic ndings of xenografts of the

immunosuppression and immunotolerance protocols. In thrombotic microangiopathic glomerulopathy at postoperative day 33
in B114 in the immunosuppression protocol, the glomerular
capillary structure was lost with apoptotic cell (asterisk) and brin
deposition (A: original magnication, 35000), mesangial matrixlysis and activated and enlarged mesangial cells (asterisk) (B:
original magnication, 34000), and activated and enlarged endothelial cells (asterisk) (C: original magnication, 36000). In reversible microangiopathic glomerulopathy in the immunotolerant
baboon (B113), swelling of endothelial cells (asterisk) was noted
in glomeruli with partial dilatation of subendothelial space and
mesangiolytic changes at postoperative day 13 (D: original
magnication, 32000; E; original magnication, 37000). However, in the immunotolerance group, these ndings recovered to
almost intact glomerular structure by postoperative day 29 (B113:
F; original magnication, 32000). The glomeruli in the graft with
unstable graft function from postoperative day 50 showed double contour of glomerular basement membrane (arrow in G),
mesangial proliferation, and effacement of foot processes of
podocytes at postoperative day 81 (B118: G; original magnication, 37000). The glomeruli in the graft with stable graft
function showed almost intact glomerular capillaries with preservation of foot process of podocytes at postoperative day 83
(B134: H; original magnication, 35000).

and CD68+ macrophage inltration, which may be associated

with AHXR, many CD3 and TIA-1+ cytotoxic T cells were also
present in the inltrate with tubulitis, peritubular capillaritis,
acute glomerulitis, and endothelialitis. These ndings suggest
that T celldependent immunologic pathways play an important
role in xenograft rejection. Recent studies also demonstrated
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Figure 5. Pathologic changes in the grafts of the immunotolerance

protocol. In reversible glomerular injury in B113, deposition of IgM
(A), C4d (C), and C5b-9 (D), but not IgG (B), occurred by postoperative day 13 (AD: original magnication, 3200), and mild
thrombotic microangiopathic glomerulopathy developed (I: periodic acid-methenamine [PAM] stain; original magnication, 3600).
Cellular rejection and arteriolar changes were not detected at
postoperative day 13 in B113 (J: hematoxylin and eosin stain;
original magnication, 3200). However, these glomerular alterations recovered to almost their structure by postoperative day 29
in B113 (K: PAM stain; original magnication, 3600). Cellular rejection, arteriolar changes, and interstitial brosis did not develop
at postoperative day 29 in B113 (L: hematoxylin and eosin stain;
original magnication, 3200). In the graft with unstable graft
function (B118), focal mesangial proliferation with double contour
of glomerular basement membrane were seen at postoperative
day 81 (M: PAM stain; original magnication, 3600). Focal interstitial brosis was noted, but cellular rejection and arterial
changes were not detected (N: hematoxylin and eosin stain;
original magnication, 3200; O: CD3 stain; original magnication,
3200; P: elastica-Masson Goldner stain; original magnication,
3600). In the graft with stable graft function (B134) at postoperative day 83, focal and weak deposition of IgM (E), C4d (G),
and C5b-9 (H) was seen, but no deposition of IgG (F) was noted
(EH: original magnication, 3200). The glomeruli showed no
obvious glomerulopathy at postoperative day 83 in B134 (Q:
PAM stain; original magnication, 3600) with no cellular rejection or vascular changes (R: hematoxylin and eosin stain; original
magnication, 3200; S: CD3 stain; original magnication, 3200;
T: elastica-Masson Goldner stain; original magnication, 3200).
POD, postoperative day.

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Figure 6. Immunopathological characterization of thrombotic

microangiopathic glomerulopathy. TUNEL+ dead cells (A and B:
original magnication, 3600), CD31+ glomerular capillaries
(C and D: original magnication, 3600), PCNA+ proliferating cells
(E and F: original magnication, 3600), tissue factor expression
(G and H: original magnication, 3600), vWF expression (I and J:
original magnication, 3600), and CD39 expression (K and L:
original magnication, 3600) in glomeruli of the immunosuppression (A, C, E, G, I, and K: B114, postoperative day 34) and immunotolerance (B, D, F, H, J, and L: B134, postoperative day 83)
baboons. In the immunosuppression protocol, thrombotic microangiopathic glomerulopathy developed at postoperative day 34 in
B114 with many TUNEL+ dead cells (arrow) (A), loss of CD31+ glomerular capillaries (C), proliferating PCNA+ cells (arrow) (E), increased
expression of tissue factor (G) and vWF (I), and decreased expression
of CD39 (K). In contrast, the glomeruli of the immunotolerance baboon (B134) at postoperative day 83 were characterized by preservation of CD31+ glomerular capillaries (D) with rare TUNEL+ dead
cells (B) and PCNA+ cells (F), no upregulation of tissue factor (H) and
vWF expression, and persistent CD39 expression (L).

the involvement of T cells in kidney xenograft rejection in miniature swine or human decay accelerating factor pigtobaboon
model and in heart xenografts in GalT-KO swinetobaboon
transplantation.28,3032 In vitro assays also showed T cell sensitization and T celldependent cytotoxic antibody production
after transplantation in recipients of the long-term immunosuppression protocol.19,33 As is seen with allotransplantation,
ACXR may be a more important and common type of xenograft
rejection, and humoral rejection might be avoided by various
strategies directed at T- and B-cell immunity.
In clinical kidney transplantation, chronic allograft glomerulopathy is a characteristic pathologic nding of chronic
rejection.34,35 Ample evidence suggests that chronic rejection of
allografts is caused by a combination of chronic immune and
nonimmune injury, and that the immune mechanism involves
both cellular and humoral rejection.34,35 In addition, in the
immunotolerance protocol in pig allokidney transplantation,
induction of unstable tolerance of kidney grafts is associated
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with late progression of chronic rejection.36,37 In the present

study, chronic xenograft glomerulopathy developed in the graft
at postoperative day 81 in B118, with late unstable graft function
in the immunotolerance protocol. The pathologic features of
this glomerulopathy are similar to those seen with chronic allograft glomerulopathy in clinical kidney allotransplantation.35
Progressive AHXR and ACXR were not detected in any graft
from baboons receiving the immunotolerance protocol. However, before transplantation, small amounts of cytotoxic antinonGal antibodies were present in most of the baboons.19
Indeed, one (B113) kidney showed mild and transient graft
dysfunction from postoperative days 7 to 40 in association
with mild thrombotic microangiopathic glomerulopathy involving deposition of IgM and complements.
We think that in this protocol chronic xenograft glomerulopathy may be associated with unstable induction of
immunotolerance, graft injury from preformed antibodies,
or persistent nonimmunologic graft injury, potentially involving the deregulation of coagulation and platelet activation
in the xenogeneic vasculature; these changes are similar to what
is observed in allokidney transplantation.36,37 Previous studies
have also reported the presence of variable amounts of preformed anti-nonGal antibodies in humans.38 Additional induction therapy to reduce the levels of preformed anti-nonGal
antibodies and to prevent initial graft injury associated with
preformed anti-nonGal antibodies may be required to ensure
long-term xenograft survival. Transgenic modication of
swine to allow overexpression of human anticoagulant and
thromboregulatory factors such as CD39 may be also further
required for long-term xenograft survival.1
Clinical application of xenotransplantation will rst require
the demonstration of its efcacy in a nonhuman primate model.
In this study, the response to GalT-KO kidney xenografts in
baboons was not controlled by our long-term immunosuppression protocol, and AHXR and ACXR developed in the grafts
by postoperative day 34. In contrast, the immunotolerance
protocol prevented AHXR, ACXR, and chronic xenograft
rejection in two of four GalT-KO kidney grafts by postoperative
days 56 and 83. Unfortunately, in this study, all the animals in the
immunotolerance protocol died of complications not associated
with rejection. We believe that improved strategies for rapid and
stable induction of xenogeneic T cell tolerance and prevention of
side effects of immunosuppression drugs, such as thrombotic
complications and infection, may be required for long-term
xenograft survival. Ongoing studies in our center are designed to
induce immunotolerance toward GalT-KO miniature swine
organs in nonhuman primates by bone marrow transplantation
and donor vascularized thymus grafting and by additional
transgenesis to protect the renal vasculature.1,39
CONCISE METHODS
Animals
Seven life-supporting GalT-KO kidney transplantations were performed in baboons (Papio anibus; Manheimer Foundation,
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for Medical Research and the Guide for the Care

and Use of Laboratory Animals prepared by the
Institute of Laboratory Animal Resources and
published by the National Institutes of Health.
All protocols were approved by the MGH Subcommittee on Research Animal Care.

Pig-to-Baboon Xenotransplantation

Figure 7. Cellular inltration within Gal knockout kidneys in baboons treated with
chronic immunosuppression or immunotolerance protocol. Cellular inltration in immunosuppression (AJ: B114, postoperative day 34) and immunotolerance (KN:
B134, postoperative day 83) protocols. Cellular inltration developed at postoperative
day 34 (B114) (A: hematoxylin and eosin stain; original magnication, 3400) in the
immunosuppression protocol with inltration of CD3+ T cells (B: original magnication, 3400) and CD68+ macrophages (C: original magnication, 3400), although with
little inltration of CD20+ B cells (D: original magnication, 3400) and NK cells (E:
original magnication, 3400). Many CD3+ (brown) T cells contained TIA-1+ (blue)
cytotoxic granules (arrow) (F: original magnication, 3900), capillaritis, acute glomerulitis, and endothelialitis developed with CD3+ T cells (arrow) in tubules (G: original
magnication, 3800), peritubular capillaries (H: original magnication, 3800), glomeruli (I: original magnication, 3800), and arteries (J: original magnication, 3800).
In contrast, only small numbers of CD3+ T cells (K: original magnication, 3200) and
CD68+ macrophages (L: original magnication, 3200), and very few CD20+ B cells
(M: original magnication, 3200) and NK cells (N: original magnication, 3200) were
present in the grafts at postoperative day 83 in B134 in the immunotolerance protocol.
Analysis of the number of inltrating CD3+ T cells, macrophages, CD20+ B cells, and
NK cells in the grafts (cells/single eld of 0.0625 mm2) indicated increased inltration
of T cells and macrophages in the grafts of the immunosuppression protocol, but not
those of the immunotolerance protocol. HPF, high-power eld; POD, postoperative day.

Homestead, FL) weighing 712 kg; GalT-KO miniature swine weighing 927 kg were used as donors.19 All GalT-KO donors were individually generated by nuclear transfer from GalT-KO broblasts from
Massachusetts General Hospital MHC-inbred miniature swine.15 All
animal care procedures were performed in accordance with the Principles of Laboratory Animal Care formulated by the National Society
232

Journal of the American Society of Nephrology

In the immunotolerance protocol, we performed 11 experimental transplants of kidney

plus vascularized thymus.19 In the present study,
however, 3 of 11 transplants were excluded because of early termination (days 4, 13, and 33)
with cessation of immunosuppression (n=2) or
cerebral infarction (n=1). The other four experimental animals were also excluded because
these animals with stable renal function died
of causes unrelated to the transplant by day 31.
The pathologic features of the grafts in these
four animals on days 16, 18, 26, and 31 were
similar to those of the grafts with long-term
survival (n=4), but the chronic period of the
grafts could not be examined. Therefore, we focused on 4 of the 11 transplants in the immunotolerance protocol that survived more than
50 days and could thus be characterized long
term. In our immunotolerance protocol, kidney
transplantation was performed together with
vascularized thymus as a thymokidney or a
vascularized thymic lobe.33,40,41 In two cases of
thymokidney transplantation (B118 and B134),
composite thymokidney grafts were prepared
from GalT-KO miniature pigs by the implantation of autologous thymic tissue under the kidney capsule 68 weeks before transplantation to
allow thymic revascularization prior to transplantation.19,42 In two cases (B113 and B135),
baboon recipients received an orthotopic kidney
and a donor-matched vascularized thymic lobe
using the procedure described in detail elsewhere.19,43 Finally, in the three cases from the
immunosuppression protocol (B114, B126, and
B131), recipients received only an orthotopic
kidney without vascularized thymic tissue.

Immunosuppression

Our immunotolerance and immunosuppression

protocols were described in detail previously.19
Immunotolerance in our baboon recipients
(n=4) was induced by thymectomy, splenectomy, and T cell depletion, rst with anti-thymocyte globulin (ATG; 50 mg/kg per
day intravenously) for 34 days (Pharmacia/Upjohn). The T cell
count was reduced to ,200 cells/mm3 with 12 mg of LoCD2/kg
intravenously (rat anti-primate CD2b, Immerge, BioTherapeutics,
Charlestown, MA). Furthermore, three of the four baboons received

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Table 2. Banff classication 2007 score of graftectomy

samples
Animal ID

Acute Rejection Score

Chronic Rejection Score

ptc

C4d

ci

ct

cg

mm

cv

B114
B126
B131

1
1
1

1
1
1

2
1
1

3a
3a
3a

3
2
3

3
2
2

2
1
0

2
1
1

0
0
0

3
2
2

0
0
0

B113
B135
B118
B134

0
0
0
0

0
0
0
0

0
0
0
0

0
0
0
0

0
0
0
0

0
0
0
0

1
0
1
0

1
0
1
0

0
0
2
0

1
1
1
0

0
0
0
0

i, mononuclear cell interstitial inammation score (i0i3); t, tubulitis score (t0t3);

g, early type of allograft glomerulitis score (g0g3); v, intimal arteritis score
(v0v3); ptc, peritubular margination of inammatory cells score (ptc0ptc3);
C4d, C4d score (C4d0C4d3); ci, interstitial brosis score (ci0ci3); ct, tubular
atrophy score (ct0ct3); cg, allograft glomerulopathy score (cg0cg3); mm,
mesangial matrix score (mm0mm3); cv, brous intimal thickening score (cv0cv3).
a
Presence of interstitial hemorrhage.

100 rads of whole-body irradiation on day 26, followed by reductions in the doses of ATG (100 mg/kg) and LoCD2 (8 mg/kg). Maintenance therapy consisted of human anti-human CD154 mAb
(ABI793, Novartis Pharma, Basel, Switzerland) administered intravenously at 2025 mg/kg on day 21 and continued every 25 days
thereafter, and mycophenolate mofetil (Roche, Nutley, NJ), which
was administered by continuous intravenous infusion at 110 mg/kg
per day from day 27. All animals, except B134, received methylprednisolone, 2 mg/kg per day intravenously (Upjohn, Kalamazoo, MI),
starting on the day of transplantation; the dose was tapered slowly
thereafter. In the immunosuppression protocol, one baboon (B126)
received the same immunotolerance induction regimen as the experimental animals. The two other animals (B114 and B131) underwent
immunosuppression protocol at our center, which included 700-cGy
thymic irradiation on day 22, anti-CD154 mAb, mycophenolate
mofetil, and steroids, but did not undergo thymectomy. All recipients
received cobra venom factor daily for the rst 2 weeks after transplantation to protect against the effects of natural anti-nonGal antibodies
and of nonspecic complement activity.

Histologic and Immunohistochemical Examination

Kidney graft samples were taken during open biopsies and from
graftectomies (Table 1). For light microscopic examination, tissue
was xed in 10% buffered formalin and embedded in parafn. Sections were examined after hematoxylin and eosin, periodic acidSchiff, Masson trichrome, periodic acid-methenamine silver, and
elastica-Masson Goldner staining. Tissues for electron microscopy
were xed in 2.5% glutaraldehyde plus 2% paraformaldehyde, postxed with 1% osmium tetroxide, and embedded in Epon 812. Ultrathin sections were stained with lead citrate. Frozen tissue sections
were stained by direct immunouorescence using uorescein isothiocyanate (FITC)-conjugated rabbit polyclonal antibody to human IgG
(1:30), IgM (1: 20), C3 (1:10), and brinogen (1:20) (all from Dako,
Carpinteria, CA); for indirect immunouorescence, antihuman C4d
mAb (Quidel, San Diego, CA), polyclonal rabbit anti-human C4d
antibody (American Research Products, Inc., Belmont, MA) and
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BASIC RESEARCH

anti-human C5b-9 mAb (Dako) were used. The C4d antibody does
not stain pig C4d, so it would not detect residual donor C4d.
The following primary antibodies were used for staining by the
standard avidin-biotin-peroxidase complex technique:28 (1) antiswine CD31 (PCAM1) mAb (Serotec, Raleigh, NC) to detect capillary
endothelium in swine grafts; (2) polyclonal rabbit anti-tissue factor
antibody (the cross-reactive antiporcine tissue factor), provided by
Professor Yale Nemerson (Mount Sinai School of Medicine, New
York, NY),44,45 to detect tissue factor on porcine activated endothelial
cells; (3) anti-pig CD39 mAb,46 to detect nucleoside triphosphate
diphosphohydrolase (a thromboregulatory factor) on porcine endothelial cells; (4) anti-human CD41 mAb (5B12: Dako) to detect baboon platelets; (5) polyclonal rabbit anti-human vWF (Dako), which
detects vWF in endothelial cells and thrombi; (6) anti-PCNA mAb
(PC10: Dako), which detects proliferating cells; (7) antiTIA-1 mAb
(granule membrane protein of 17 kD; Coulter Immunology, Hialeah,
FL) to detect cytotoxic granule protein in T and NK cells; and (8)
polyclonal rabbit anti-human CD3 antibody (A0452; Dako), antihuman CD20 mAb (B cells, L26; Dako), anti-human CD68 mAb
(macrophages, KP-1; Dako), anti-human NKB1 mAb (NK cells,
DX9; BD Biosciences, San Jose, CA), and anti-human CD56 mAb
(neural cell adhesion molecules/NK cells, 1B6; Nitirei), which were
used to determine the phenotype of inltrating cells.
To detect platelet-brin thrombi in xenografts, two-color immunohistochemistry for CD41 (Texas Red) and brinogen (FITC) was
performed using frozen sections. The relationship between CD41+
platelet aggregation and the deposition of immunoglobulin or
complement was assessed in frozen sections using two-color immunohistochemistry for CD41 (Texas Red) and IgM (FITC) or C4d
(FITC). To detect cytotoxic T cells in xenografts, two-color immunohistochemical staining against TIA-1 (alkaline phosphatase, blue) and
CD3 (3, 39-diaminobenzidine, tetrahydrochloride, brown) was performed in parafn-embedded sections. In histologic sections, fragmented nuclear DNA associated with apoptosis was labeled by the
terminal deoxynucleotidyl transferasemediated TUNEL method.31

Quantication of Histologic Findings

In each kidney sample, 40 randomly selected interstitial elds

(0.065 mm2) or 40 glomerular cross-sections were assessed at a magnication of 3400, without prior knowledge of the clinical or histologic
ndings, for the following measures: (1) thrombotic microangiopathic glomerulopathy: the mean semi-quantitative severity score
of glomerulopathy, per glomerular cross-section in periodic acidSchiffstained sections (scores 03: 0, no glomerulopathy; 1, segmental
and mild glomerulopathy; 2, segmental and moderate glomerulopathy; 3, global and severe glomerulopathy); (2) interstitial hemorrhage: the mean semiquantitative severity score of interstitial
hemorrhage, per interstitial eld in hematoxylin and eosinstained
sections (scores 0 to 3: 0, no hemorrhage; 1, focal and mild hemorrhage; 2, focal and moderate hemorrhage; 3, diffuse and marked
hemorrhage); (3) evaluation of deposition of IgM and IgG, complement (C4d and C5b-9), and CD41, and expression of tissue factor,
vWF, CD39: mean semiquantitative staining score of IgM, IgG, C4d,
C5b-9, CD41, tissue factor, vWF, or CD39, respectively, per glomerular cross-section (scores 04: 0, no localized increase in staining; 1,
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little increase in staining [deposition in two or three glomerular capillaries]; 2, moderate increase in staining [involving ,25% of the glomerular capillaries]; 3, intense increase in staining [involving ,50% of
capillaries]; and 4, maximal increase in staining [50% capillary involvement]);28 (5) glomerular capillaries: the mean number of glomerular capillary lumina surrounded by CD31+ cells, per glomerular
cross-section; (6) the frequency of PCNA+ cells in glomeruli: the
mean number of PCNA+ cells per glomerular cross-section; and
(7) the number of CD3+, CD68+, CD20+, and NK cells in the interstitium: the mean number of CD3+, CD68+, CD20+, and NKB-1+
cells in selected interstitial elds (0.065 mm2). Correlations between
the deposition of C4d and the severity of glomerulopathy or CD41
deposition were computed and analyzed using a Pearson test. In the
graftectomy samples, acute and chronic rejection scores were evaluated using Banff classication 2007.20

Renal Function
Arterial blood sampling was performed twice daily to measure serum
creatinine and blood BUN. The endpoint for renal xenograft survival
was rejection, as indicated by plasma creatinine levels .9 mg/dl or
by severe deterioration of clinical condition due to uremia (BUN
.100 mg/dl).

ACKNOWLEDGMENTS
The authors thank Drs. John and Isabel Hanekamp and Dr. Leo
Buhler for critical review of the manuscript.
This work was supported in part by National Institutes of Health
Program Project 1PO1 A145897 and 5PO1-A145897 and by a
Sponsored Research Agreement between Immerge BioTherapeutics,
Inc. and the Massachusetts General Hospital. A.S. was a recipient of a
grant from the Japanese Society for the Promotion of Science, Grantin-Aid for Scientic Research (C20591900). The authors thank Novartis
Pharma AG (Basel, Switzerland) for generously providing anti-CD154
mAb and Immerge BioTherapeutics Inc. (Cambridge, MA) for providing LoCD2b mAb and cobra venom factor.

DISCLOSURES
None.

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