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Stable a- and b-Islet Cell Function After ToleranceInduction to Pancreatic Islet Allografts inDiabetic Primates
Juan L. Contrerasa, Stacie Jenkinsa,Devin E. Eckhoffa, William J. Hubbarda,Andrew Lobashevskya, Guadalupe Bilbaob,Francis T. Thomasa, David M. Neville Jra andJudith M. Thomasa,*
aThe Transplant Center, Department of Surgery, and bGeneTherapy Center, University of Alabama at Birmingham,Birmingham, Alabama 35294, USAcThe Laboratory of Molecular Biology, National Instituteof Mental Health, Bethesda, Maryland 20800, USA*Corresponding author: Judith M. Thomas, [email protected]
Pancreatic islet transplantation (PIT) is an attractivealternative for type 1 diabetic patients. PIT is not yetan effective clinical reality due in part to early loss offunctional islet mass. In addition, current immuno-suppressive drugs have toxic effects on islets andincrease the risk of morbidity and mortality. Preciseand durable a- and b-cell function is essential for thesuccess of PIT. Therefore, it is important to establishwhether PIT can produce adequate long-term meta-bolic control, especially in the absence of chronicimmunosuppressive therapy (CIT). In the presentstudy, the stability of functional a- and b-cell massand metabolic function was assessed in strepto-zotocin (STZ)-induced diabetic primates following PITin the absence of CIT. Diabetes was induced in rhesusmacaques with STZ, 140 mg/kg. Hyperglycemia wasreversed rapidly by PIT coupled with a 14-day toler-ance induction protocol based on F(Ab)2-IT and DSG(n = 7). Two diabetic animals received the toleranceinduction protocol without PIT. Acute rejection waspresented in three animals at 70, 353 and 353 dayspost transplant in the tolerance induction protocol,whereas the controls [F(Ab)2-IT or DSG alone] showedearly 10-day function but all lost islet function bydays 15–70. One recipient [F(Ab)2-IT or DSG] diedeuglycemic after a surgical procedure on day 187. At2 years, three animals studied had a normal FIMevaluated by oral glucose tolerance test, mixed mealtest, acute insulin response to glucose, glucosedisposal rate, and hyperinsulinemic hypoglycemicclamp. PIT in STZ-induced diabetic primates resultedin restoration of normal a- and b-cell function. Oper-ational tolerance induction was achieved with onlyperitransplant administration of F(Ab)2-IT and DSGsparing the animals from chronic exposure of dia-betogenic immunosuppressive drugs. These resultsoffer an exciting new potential for treatment oftype 1 diabetes mellitus.
Key words: Islet mass, islet transplantation, primates,tolerance
Received 21 June 2002, revised and accepted forpublication 23 August 2002
Introduction
Pancreatic islet transplantation (PIT) in a promising alter-
native to whole-organ pancreas transplant for type 1
insulin-dependent diabetes mellitus (IDDM) (1,2), but
wide clinical application of PIT is hindered by low rates of
success. For example, in the most successful clinical
experience with PIT, two or more islet infusions were
necessary to achieve sufficient functional islet mass
(FIM) for establishing normoglycemia without exogenous
insulin administration (3,4). While islet re-transplantation
is effective, it lacks cost-effectiveness and is constrained
by the shortfall of donor pancreatic tissue. Thus, new
strategies for improving durable FIM will be instrumental
in facilitating PIT as a cure for IDDM. The mechanisms that
underlie post-transplant loss of FIM are poorly understood.
Rejection, recurrence of autoimmunity, insufficient func-
tional b-cell mass, poor engraftment, and direct toxicity of
the implanted b-cells by immunosuppressive drugs are all
postulated to play a role in graft failure (1,2). Prednisone,
cyclosporine, azathioprine, and tacrolimus have all been
shown to cause modulation and/or impairment of insulin
synthesis and secretion (5). After the peri-operative period,
immunosuppressive drugs are taken orally, resulting in high
intraportal drug concentrations and potential adverse
effects on intrahepatic islet transplant function (5,6).
The ideal approach for diabetic therapy would involve peri-
transplant induction of tolerance to islet grafts to promote
stable, long-term islet function without chronic immuno-
suppressive therapy (CIT). Studies from different groups
suggest that tolerance induction is possible in nonhuman
primates without CIT (7,8). Our group reported stable
kidney transplant function for years in operationally tolerant
rhesus monkeys without evidence of acute or chronic
rejection (9–12). We also demonstrated stable reversal of
spontaneous diabetes in primates by islet xenografts
without CIT and more recently in STZ-induced diabetic
recipients (13,14). This tolerance has been achieved
by employing a short peritransplant treatment combination
of anti-CD3 receptor-specific diphtheria-based IT and
American Journal of Transplantation 2003; 3: 128--138 Copyright # Blackwell Munksgaard 2003Blackwell Munksgaard
ISSN 1600-6135
128
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15-deoxyspergualin (DSG). IT transiently depletes naive and
memory T cells in blood and lymph-node compartments,
while DSG concomitantly blocks pro-inflammatory cytokine
production and maturation of dendritic cells by inhibiting
nuclear translocation of NF-kB (9–12).
Individuals with long-standing type 1 diabetes mellitus
have impaired hypoglycemic hormonal counter-regulation,
which puts them at risk of severe hypoglycemia (15).
Pancreas transplantation has been demonstrated to
normalize glucagon response and symptom recognition
during hypoglycemia for up to 19 years after transplantation
(16). However, no study has yet examined hypoglycemia
counter-regulation after intraportal islet transplantation in
diabetic nonhuman primates. This is a critical issue for
patients and physicians who use PIT as a means of treating
recurrent, life-treating hypoglycemia.
Precise and durable glycemic control is essential for the
success of islet transplantation. Therefore, it is important
to establish whether islet transplantation can produce
adequate long-term metabolic control, especially in the
absence of CIT. In the present study, the stability of
functional a- and b-islet cell mass and metabolic functions
were assessed in STZ-induced diabetic nonhuman primates
following IT plus DSG tolerance induction in the absence of
CIT. Detailed observations of other experiments of survival,
immunosuppressive therapy and immune function during
and following tolerance induction have been covered else-
where (17), and will not be discussed here.
Materials and Methods
Animals
Normal male rhesus monkeys (Macaca mulatta), 3.0–3.8 kg, were obtained
from Covance Research Products (Alice, Texas). Animals had negative
titers for anti-diphtheria toxin antibodies by ELISA as described (11,12).
All procedures were performed in accordance with the National Institute of
Health Guide for the Care and Use of Primates Guidelines of the Institutional
Animal Care and Use Committee. Ketamine (10 mg/kg, Fort Dodge Labora-
tories, Fort Dodge, IA) and acepromazine (1 mg/kg, Vedco laboratories,
St. Joseph, MO) was used i.m. for routine handling of the animals.
Induction of diabetes
To induce diabetes, normal animals were treated 2–4 weeks before PIT with
an i.v. bolus of STZ (Sigma, St. Louis, MO) at 140 mg/kg mixed immediately
before administration with 5 mL citrate buffer. As previously published,
a single large dose of STZ reliably induces IDDM in primates (18).
Donor and recipient combinations
All donor and recipient combinations were selected to have multiple donor
MHC Class I and II mismatches by molecular typing as previously
described (19,20).
Preparation of islets
Pancreata were removed from 5–6.5 kg normal rhesus monkeys as
described (17).The islets were separated by a semi-automated technique
and purified using a COBE discontinuous flow centrifuge (Lakewood, CO).
The cells were counted manually and sized using a formula to calculate
islet number and islet equivalents (IEQ) based on a 150-mm diameter.
Dithizone staining was used to monitor islet purity and acridine orange/
ethidium bromide was employed to assess islet viability. Only islet
preparations with purity and viability >90% were used. The isolated islets
were cultured overnight in CRML 1066 (Mediatech, Indianapolis, IN)
containing 10% FBS at 25–28 �C in 95% O2, 5% CO2.
Pancreas islet transplantation
A mean of 23 363� 2157 IEQ/kg per recipient was infused by gravity flow
as described (17). No significant hemodynamic or portal vein pressure
changes were observed. One donor was used for two recipients in two
occasions and one donor per recipient in the rest.
Immunosuppressive therapy for tolerance induction
Therapy was initiated on the day of transplant and discontinued on day 15 post
transplant. All animals received methylprednisolone (MP), 7 mg/kg/day/i.v.
4 h before the transplant, 3.5 mg/kg/day/i.v. on day 1 and 0.35mg/kg/day/i.v.
on day 2. The IT used in this study was a chemical conjugate of CRM9
and the F(Ab)2 fraction of anti-rhesus CD3 (FN18) IgG1 as previously
described (12,17). The IT was administered as a bolus of 100mg/kg/day/i.v.
2 h before the transplant and 100 mg/kg/day/i.v on day 1. DSG (NKT01,
Brystol-Myers, Princeton, New Jersey, Nippon Kayaku, Tokyo, Japan)
was administered as a bolus of 2.5 mg/kg/day/i.v., beginning 4 h
pretransplant and continuing daily through day 14. No immunosuppressive
agents were given after day 15. In order to evaluate the effect of the
tolerance induction protocol on the natural history of the IDDM, two
animals were induced with STZ and received IT and DSG without islet
cell infusion.
Post-surgical care
Recipients received prophylactic antibiotic therapy with Cefazolin (Eli Lilly,
Indianapolis, IN) 12.5 mg/kg/BID on days 0–6 and Doxycycline (Pfizer,
Evanson, IL) at 1.5 mg/kg/day on days 7–21. Oral Ensure Plus,
50–100 mL/day provided supplemental nutritional support for 2 weeks
post transplant. Buphenorphine hydrochloride (0.05 mg/kg/BID/i.m. Reckit
Colman Pharmaceuticals, Richmond, VA) was used for analgesia after
surgery. No exogenous insulin was given at any time after the transplant.
No exceptional safeguards against infection were introduced beyond
standard AALAC primate care.
Monitoring b-islet cell transplant function
Non-fasting blood glucose was monitored daily at 0800–0900 with a gluco-
meter (Accu-chek, Roche, Indianapolis, IN), and weekly by glucose oxidase
using an automated analyzer (University of Alabama at Birmingham
Outreach Laboratory). Ketamine hydrochloride was used for anesthesia
(10 mg/kg/i.m. initial dose, 5 mg/kg/i.m. subsequent doses) and was kept
as low as possible during all metabolic studies.
Fasting plasma glucose and responses to intravenous glucose load were
determined in all animals before the treatment and transplant. These tests
were performed following an 18-h overnight fast, with a constant antece-
dent diet. Intravenous glucose tolerance tests (IVGTT) were carried out as
previously described (21). Briefly, intravenous catheters were placed in
both legs. The leg from which samples were obtained was warmed to
assure arterialization of venous samples. Three baseline blood samples
were collected at 5-min intervals. Then 0.5 g/kg of dextrose 50% was
infused intravenously over a 30-s period, and blood samples were with-
drawn at 1, 2, 5, 7, 10, 12, 15, 20, 30, 45, and 60 min relative to glucose
Stable Islet Mass After Tolerance
American Journal of Transplantation 2003; 3: 128--138 129
Page 3
injection. Samples were collected in glass tubes containing heparin, placed
on ice, and centrifuged within 10 min. Plasma was stored at �20 �C until
assayed for glucose and insulin. Plasma insulin levels were measured by
a double antibody method by the University of Alabama at Birmingham
Hospital Outreach Laboratory using human insulin as a standard. Rhesus
monkey insulin has been shown to be identical to human insulin (22).
The oral glucose tolerance test was performed after an overnight fast.
Dextrose, 1 g/kg in 20–30 cc of water was given by feeding tube, and
blood samples for glucose determination were obtained at 120 min (3).
For the mixed meal tolerance test (3), Ensure was given at 30 mL/kg via
feeding tube. Blood samples were taken at 0 and 90 min for C-peptide level
assessment by The University of Alabama at Birmingham Hospital
Outreach Laboratory.
The glucose disappearance constant (kg) was calculated from the IVGTT
according to the following formula: kg¼70/t½, where t½¼ number of min
to fall 50% glucose concentration from its 1-min level (23). The acute
insulin response to glucose (AIRglucose) was calculated as the mean of
the post-stimulus (dextrose, 0.5 g/kg/i.v.) samples obtained at 1, 3 and
5 min after i.v. glucose injection minus the mean of the three pre-stimulus
samples (23,24).
A transhepatic insulin release was performed in two recipients. After
a midline incision, catheters were placed on the suprahepatic veins, portal
vein and saphenous vein. After a dextrose bolus (1 g/kg), insulin levels
were measured at 1, 3, 5, 7 and 10 min to evaluate insulin secretion from
the liver vs. the pancreas.
Monitoring a-islet cell transplant function
Long-term survivors, at least 2 years post transplant, underwent a 100-min
stepped hypoglycemic clamp along with control diabetic and normal
control rhesus monkeys. Briefly, after a 12-h fast, under light ketamine/
acepromazine anesthesia, a 24G intravenous catheter was placed in the
right saphenous vein for insulin and glucose infusion as previously
described (25). Venous blood was sampled from a 20G catheter placed in
the contralateral saphenous vein. Blood glucose determination was
obtained every 5 min with a glucometer. After a 15-min equilibrium period,
recombinant human insulin (Humulin, Lilly, Indianapolis, IN) was infused at
2 mU/kg/min. A variable rate 20% glucose infusion was used to maintain
blood glucose concentrations at the target glucose plateaus of 60, 55, 40
and 35. Arginine (70 mg/kg, as 10% arginine HCL; KabiVitrum, Clayton, NC)
was given i.v. 30 min after the initiation of the study. Blood samples were
obtained during the basal period and at 25-min intervals for determination
of glucagon levels by the University of Alabama at Birmingham Hospital
Outreach Laboratory. Heart rate and blood pressure were monitored
throughout the study.
Statistical analysis
Results are expressed as a mean�SEM. A double-sided t-test was used
for statistical analysis. Statistical significance was scored at p<0.05.
Results
The mean islet equivalent (IEQ) recovered per pancreas
was 112 066� 46 149 (11 421� 4003 IEQ/g pancreas).
Mean purity was 95.5� 3.9%, and mean viability was
94.5� 3.8% after 24 h. Endotoxin levels in the isolation
media were low, mean 0.5� 0.3 EU/mL. All animals treated
with STZ exhibited fasting glucose levels >150mg/dL, non-
fasting glucose levels >280 mg/dL, almost undetectable
stimulated C-peptide levels (<0.6 ng/mL, compared with
normal control monkeys, range 0.9–4.1 ng/mL), and abnor-
mal glucose disposal rate following IVGTT (kg <0.7%/min
vs. >3.5%/min for normal controls). All STZ-induced
diabetics required 2–4 units of human insulin twice a day
to maintain nonfasting glucose levels between 250 and
400 mg/dL.
On the day after transplantation, all PIT recipients
achieved normoglycemia without exogenous insulin
administration. All the recipients treated with the
complete tolerance induction protocol (ITþDSG) had pro-
longed graft survival without CIT (Table 1, Figure 1). One
recipient died euglycemic at 187 days from complications
following a transhepatic insulin release study (see below)
and one presented acute rejection at 70 days. All five
long-term, euglycemic recipients were subjected to
strong immune challenge to assess general immunocom-
petence. After a series of third-party skin allografting,
pneumococcal and hepatitis B vaccination, and DNCB
administration� 3, 2/5 long-term recipients began to
require exogenous insulin administration at 353 and
560 days post-transplant, respectively. These were scored
as rejections. In the other three remaining long-term recipi-
ents, operation tolerance was not perturbed by immune
stimulation, and all three are currently euglycemic at
>1092,>1035 and >946 days following a single infusion
of MHC mismatched allogeneic islets without CIT or
exogenous insulin.
In contrast to the recipients treated with ITþDSG, recipi-
ents given IT or DSG alone, failed to become long-term
survivors. These results are consistent with a synergy
between IT and DSG, previously reported for operational
tolerance induction in kidney transplant recipients (9–12).
Additionally, no changes in the glycemic parameters or
insulin requirement were observed after 1.5 years of
Table 1: Allogeneic PIT survival in STZ-induced diabetic primates
I.D. Treatment Graft-survival (days)
97D027 F(Ab)2-ITþDSG 560*
97D001 F(Ab)2-ITþDSG 353*
97D004 F(Ab)2-ITþDSG 187***
97D208 F(Ab)2-ITþDSG >985
97D164 F(Ab)2-ITþDSG 70**
97D291 F(Ab)2-ITþDSG >928
97D207 F(Ab)2-ITþDSG >839
97D052 DSG 16**
97D080 DSG 15**
97D480 F(Ab)2-IT 70**
97D215 F(Ab)2-IT 23**
*Hyperglycemia after a strong immune challenge (third-party skin
graft, pneumococcal and hepatitis B vaccination and DNCB
administration� 3).
**Acute rejection.
***Died euglycemic after a surgical complication.
Contreras et al.
130 American Journal of Transplantation 2003; 3: 128--138
Page 4
follow-up in animals that received the complete tolerance
induction protocol ITþDSG without PIT (Figure 1).
To examine the islet function after transplantation, all PIT
recipients underwent a comprehensive series of meta-
bolic tests. The results of the oral glucose tolerance
tests, performed at days 10, 365 and 750 post transplant,
are shown in Figure 2. No significant difference in fasting
glucose levels and glucose at 120 min after an oral glucose
load was observed 10 days after the transplant in recipi-
ents treated with IT and DSG or DSG alone, compared
with normal primates from our colony. Animals that
received IT alone presented higher fasting glucose levels
(107.5� 6.36 mg/dL) and higher glucose at 120 min
(130� 8.48 mg/dL) after glucose infusion compared with
those given ITþDSG (65.71� 6.57 and 81.28� 9.01 mg/dL)
or DSG alone (70� 2.8 and 84� 2.82 mg/dL). These
results are in accordance with previous studies in
small animal models that demonstrated a cytoprotective
effect of DSG on islet survival after transplantation
(26,27).
Abnormal IVGTT values were demonstrated in animals
that received the complete protocol without PIT. No sig-
nificant difference in fasting glucose levels or glucose at
120 min were observed 1 and 2 years post transplant in
the long-term survivors compared with values obtained
10 days post transplant. Abnormal values were observed
after acute rejection in animals that received IT or DSG
alone (Figure 2). Most animals with rejection became ill on
long-term insulin therapy were euthanized and not avail-
able for metabolic testing further that a year.
DSG + PIT
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Figure 1: Non-fasting glucose levels in diabetic primates. Non-fasting glucose levels were determined as described in Methods.
(A) Animals received the complete tolerance protocol (ITþDSG, n¼7). (B) IT alone (n¼2). (C) DSG (n¼2) or ITþDSG (n¼ 2) without PIT.
Stable Islet Mass After Tolerance
American Journal of Transplantation 2003; 3: 128--138 131
Page 5
Mixed meal tests (Figure 3) showed no significant differ-
ence in C-peptide levels and glucose at 120 min before
and after Ensure administration in long-term PIT survivors
compared with normal controls. In contrast, failure of
C-peptide to increase in serum after the challenge was
observed in PIT recipients that received either IT or DSG
alone. These results confirm stable, long-term FIM with
operational tolerance in the absence of CIT in the ITþDSG
group given PIT.
Previous studies demonstrated the glucose disposal rate
(kg) to be a sensitive marker to monitor endocrine function
in pancreas transplant recipients and to evaluate the sur-
viving islet mass after islet transplantation (23,28–30).
Prior to PIT, all diabetic recipients presented an abnormal
kg (<0.7%/min), compared with normal primates from
our colony (>3.5%/min, Figure 4). Ten days post-PIT,
normalization of the kg was observed in animals
treated with ITþDSG treatment or with DSG alone. As
observed in the oral glucose tolerance test, a significant
increase in kg values were seen also in animals treated
with IT alone, but it was significantly lower compared to
normal animals or animals that received DSG alone
(Figure 4). No difference in the glucose disposal rate was
found in the long-term survivors 1 and 2 years after the
transplant compared with values obtained on day 10,
demonstrating long-term stability of the functional islet
mass after transplantation. No improvements in kg values
were measured in animals treated with ITþDSG without
PIT up to 12 months.
The islet mass was assessed at 1 year post transplant by
AIRglucose. As demonstrated in Figure 5, no increases in
AIRglucose were found after PIT in monkeys treated with
either IT or DSG alone or the tolerance induction protocol
without PIT. In contrast, the long-term tolerant group
showed a high AIRglucose that was similar to AIRglucose
values obtained from normal controls. In addition,
higher AIRglucose was demonstrated in two recipients
180 days after the transplant in the suprahepatic veins
(20.8� 1.97 mIU/mL) compared with values obtained in
the portal vein (8.55� 2.61mIU/mL) or a peripheral vein
(13.15� 0.91mIU/mL), indicating that essentially all of the
insulin response to glucose derived from the intrahepatic
transplanted islets, not from the native pancreas.
Table 2 shows the serial weight changes of control and
transplanted animals as well as normal (untreated) rhesus
macaques of the same shipment group, that had
equivalent initial weights and were followed for the
same time period. The recipients with long-term islet
graft survival and euglycemia showed a 6–22% weight
gain at 180 days, a 33–55% weight gain at 365 days, and
152–162% at 750 days. In contrast, all animals that
rejected their islet grafts and were treated with exogen-
ous insulin had no significant weight gain at 365 days. No
significant difference in weight gain was observed
between long-term survivors compared with age-matched
normal male rhesus macaques (13–20% weight gain
at 180 days, 37–44% weight gain at 360, and 150–162%
at 750 days).
Day 10
0
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NormalMonkeys
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F(Ab) -IT+ DSG
2 NormalMonkeys
Figure 2: Oral glucose tolerance test in STZ-induced diabetic
primates after PIT. Fasting blood glucose and glucose at 120 min
after an oral glucose load were assessed as described in Methods
at 10 and 365 days after PIT. Only long-term survivors (4/7) in the
group that received the complete tolerance protocol (ITþDSG)
were included in the 365 days analysis and 3/7 in the 750 days.
Two animals induced with STZ received ITþDSG without PIT (no
islet infusion). Results are expressed as mean�SEM.
Contreras et al.
132 American Journal of Transplantation 2003; 3: 128--138
Page 6
Next we examined the stability of a-cell function after intra-
portal transplantation. Three groups of experimental animals
underwent a 100-min stepped hypoglycemic, hyperin-
sulinemic clamp (Figure 6). No increment in serum glucagon
was observed during the study in STZ-induced animals with-
out PIT. In contrast, a significant increase in glucagon was
observed in long-term survivors with a maximal increase of
the glucagon response from baseline to peak >50 pg/mL
following arginine administration. No significant difference
in glucagon release was demonstrated between long-term
survivors and normal monkeys from our colony. These
results confirm stable functional a-cell mass after intraportal
islet transplantation in the long-term tolerant group.
Discussion
Previous studies have demonstrated that intraportal pan-
creatic islet transplantation can restore glucose homeo-
stasis in diabetic nonhuman primates and humans (1–4,
14,17,24,31–36). In this study, we performed a compre-
hensive metabolic analysis in STZ-induced diabetic
nonhuman primates, in which tolerance was induced to
single donor allogeneic islet transplants. The results of
alpha and beta cell islet function studies indicate that
long-term, stable metabolic function can be fully restored
for years in PIT recipients in which tolerance is achieved in
the absence of CIT.
0
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eptid
e(n
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Day 750C
Figure 3: Mixed meal test in STZ-induced diabetic primates after PIT. C-peptide levels and serum glucose levels after an oral Ensure
load were assessed 1 year after PIT, as described in Methods. Only long-term survivors (4/7) in the group that received the complete
tolerance protocol (ITþDSG) were included in the analysis at 365 days, 3/7 in the 750 days. Two animals induced with STZ received
ITþDSG without PIT (no islet infusion). Results are expressed as mean�SEM.
Stable Islet Mass After Tolerance
American Journal of Transplantation 2003; 3: 128--138 133
Page 7
This study provides the first documentation of a-cell func-
tion after intraportal islet transplantation. Our data showed
that in animals given STZ without islet transplantation,
there was absence of glucagon release during a hyper-
insulinemic, hypoglycemic clamp (Figure 6). In contrast,
restoration of glucagon responses to hypoglycemia was
observed after arginine administration in the long-term
survivors at 2 years post transplant, and these responses
were not significantly different from those of normal
cohorts.
The absolute mass of transplanted islets has been iden-
tified as a critical determinant of success in PIT. However,
it is not possible to determine the actual number of islets
that implant in the liver and survive the transplantation
process. Previous studies validated that in vivo b-cell func-
tion tests give an accurate reflection of changes in func-
tional b-cell mass (28–30,37). The use of AIRglucose and kg
reflects pancreatic insulin content and functional b-cell
mass. Glucose-induced arginine potentiated insulin
release does not appear to be a significantly better
measure of engraftment than either arginine-or glucose-
induced acute insulin release in the fasting state (23).
Similar kg and AIRglucose values were observed in PIT
recipients compared with normal primates from our col-
ony (Figures 4 and 5). No changes in FIM were observed
in two control STZ-induced diabetic recipients, which
received the induction protocol without islet transplant-
ation. Detailed serial studies have demonstrated a normal
stable and durable FIM for at least 2 years follow-up in the
current rhesus macaque recipients receiving incompatible
islets from a single donor pancreas. The normal oral glu-
cose tolerance results that were obtained in the tolerant
animals (and not in non-tolerant animals) are physiolo-
gically important. Numerous diabetologists have empha-
sized that the oral route of glucose administration is a
physiological and sensitive testing system, whereas the
intravenous route is non-physiological and has been
shown to be less sensitive, since it bypasses the gastro-
intestinal (incretin) mechanisms involved in glucose regu-
lation (38).
In current practice, islet allografts in type I IDDM diabetic
recipients are exposed to deleterious effects of chronic
immunosuppressive drugs (5,6), which have a detrimental
effect on islet insulin synthesis/secretion and insulin
action (6). Reduced functional insulin secretory reserve
has also been reported after whole-organ pancreas trans-
plantation. The immunosuppressive regimen may in part
be responsible for the observed metabolic alterations,
considering that a marked reduction in insulin secretory
reserve has also been observed in nondiabetic kidney
transplant recipients (23,37). In this context, intrahepatic
islets are at risk for exposure to high concentrations of
immunosuppressive drugs present in portal blood when
these medications are administered orally. Our results in
this preclinical study on single donor intrahepatic
islet allograft survival in nonhuman primates show that,
in the absence of CIT, the non-rejectors showed no sig-
nificant change in FIM for at least 2 years after transplant-
ation. Whether the absence of CIT, the rejection-free
tolerant state, or both factors have contributed to this
outcome is uncertain.
0
5
10
15
20
25
30
35
40
45
50
Acu
teIn
sulin
Res
pons
eG
luco
se
F(Ab)2-IT+ DSG
DSG F(Ab)2-IT No IsletInfusion
NormalMonkeys
Day 365A
F(Ab)2-IT+ DSG
NormalMonkeys
Day 750
0
5
10
15
20
25
30
35
40
45
50
Acu
teIn
sulin
Res
pons
eG
luco
se
B
(IU
/ml)
µ(
IU/m
l)µ
Figure 4: Acute insulin response to glucose in STZ-induced
diabetic primates after PIT. One year after PIT (A), the acute
insulin response to glucose was calculated as the mean of the
post-stimulus (dextrose, 0.5 g/kg/i.v.) samples obtained at 1, 3 and
5 min after i.v. glucose injection minus the mean of the three pre-
stimulus. Only long-term survivors (4/7) in the group that received
the complete tolerance protocol (ITþDSG) were included in the
365 days analysis, 3/7 in the 750 days (B). Two animals induced
with STZ received ITþDSG without PIT (no islet infusion). Results
are expressed as mean�SEM.
Contreras et al.
134 American Journal of Transplantation 2003; 3: 128--138
Page 8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
F(Ab)2-IT+ DSG
DSG F(Ab)2-IT No IsletInfusion
NormalMonkeys
Kg
(%/M
inut
e)
Pre-Treatment
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
DSG F(Ab)2-IT No IsletInfusion
NormalMonkeys
Kg
(%/M
inut
e)
Day 15 Post-transplant
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
F(Ab)2-IT+ DSG
DSG F(Ab)2-IT No IsletInfusion
NormalMonkeys
Kg
(%/M
inut
e)
Day 365 Post-transplant*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
F(Ab)2-IT+ DSG
NormalMonkeys
Kg
(%/M
inut
e)
Day 750 Post-transplant*
A B
C D
F(Ab)2-IT+ DSG
Figure 5: Glucose disposal rate (kg) in STZ-induced diabetic primates before (A) and after PIT (B, C, D). kg was calculated from the
IVGTT as described in Methods. Only long-term survivors (4/7) in the group that received the complete tolerance protocol (ITþDSG) were
included in the 365 days analysis and 3/7 in the 750. Two animals induced with STZ received ITþDSG without PIT (No islet infusion).
Results are expressed as mean�SEM.
Table 2: Weights of PIT recipients before and after the transplanty
Day post-transplant
I.D. Treatment 60y 180y 365y 750y
97D027* F(Ab)2-ITþDSG 110 116.66 133.33 114.22z
97D001* F(Ab)2-ITþDSG 106.66 113.33 140 –
97D004*** F(Ab)2-ITþDSG 106.45 – – –
97D208 F(Ab)2-ITþDSG 103.33 106.66 133.33 152.44
97D164** F(Ab)2-ITþDSG 106.66 – – –
97D291 F(Ab)2-ITþDSG 107.40 114.81 155.55 162.89
97D207 F(Ab)2-ITþDSG 106.45 122.58 138.70 158.21
97D052** DSG 96.55z 103.44z – –
97D080** DSG 100z 100z – –
97D480** F(Ab)2-IT 106.66 103.33z – –
97D215** F(Ab)2-IT 89.65z 93.10z – –
97D324 ITþDSG no PIT 102.63z 100z 105.26z –
97D425 ITþDSG no PIT 97.36z 94.73z 110.52z 112.43z
AJG Normal rhesus 108.10 113.51 137.83 150.32
T4N Normal rhesus 114.70 120.58 144.11 162.41
yPercentage of weight relative to pretransplant values.
*Hyperglycemia after DNCB administration.
**Acute rejection.
***Died euglycemic after a surgical complication.zExogenous insulin therapy.
Stable Islet Mass After Tolerance
American Journal of Transplantation 2003; 3: 128--138 135
Page 9
Immunologic causes of pancreatic islet loss are an obs-
tacle to achieving insulin independence in islet allograft
recipients (1,2). Although anti-CD154 (5c8) or CTL4IgG
allowed significant prolongation of islet survival after PIT
in baboons and rhesus monkeys (33–35), a clear loss of
FIM was seen in association with rejection episodes. Our
results suggest that induction of stable tolerance can
obviate loss of FIM related to immunological events. If
so, achieving clinical tolerance could facilitate broader
application of PIT.
Our approach to tolerance induction, which employs
transient, profound pan T-cell depletion, might evoke clin-
ical concerns about generalized immune incompetence.
However, after brief peritransplant immunosuppressive
treatment with IT and DSG, the PIT recipients have not
experienced any apparent immunological compromise,
such as infections, neoplasia, or autoimmunity resulting
from the transient T-cell ablation (39). The peripheral and
lymph node T-cell recovery from a nadir of <1% of
pretreatment levels (54.3� 11.2%) to approximately
30% at 1 month and full recovery with normal phenotypic
appearance within 6–12 months post treatment (17).
Additionally, we have documented normal post-transplant
responses in tolerant PIT recipients to pneumococcal and
hepatitis B vaccinations (17). Kidney transplant recipients
treated with the same tolerance induction protocol are
capable of rejecting third-party skin transplants in less
than 10 days and produce a similar humoral immune
response to exogenous antigens compared to normal
monkeys (11,12). Despite the presence of multiple
MHC mismatches with the donor and the absence of
any maintenance immunosuppression, PIT recipients did
not develop either IgG or IgM positive-flow cross-
matches during follow-up, a finding that suggests B-cell
tolerance (data not shown, Hubbard WJ, manuscript in
preparation).
A hallmark of allograft tolerance induction after IT and DSG
administration in nonhuman primate kidney transplant
recipients is immune deviation associated with high levels
of plasma IL-10 and IL-4 and low levels of IFN-g(11). Our
recent studies reveal a significant association between
sustained type 2 cytokines (IL-10 and IL-4) and indefinite
tolerance in this model (Hutchings A et al., manuscript in
preparation). Compared with normal controls and
nontransplanted STZ-treated diabetics, the long-term PIT
survivors consistently exhibited a significant and sustained
increase in IL-10 and IL-4 levels (17). In contrast to the
indefinite survivors, the animal that lost islet function at
1 year post transplant, following vaccination challenges,
exhibited low IL-4 and IL-10 levels. Also, the animal that
lost islet function at 560 days after repeated DNCB
challenges reversed prominent type-2 cytokine deviation
to normal levels (data not shown). These observations
suggest that type-2 immune bias, possibly related to
regulatory cell activity, may have a role in the stability
of tolerance after T-cell reconstitution in nonhuman
primates.
In conclusion, PIT in STZ-induced IDDM resulted in
restoration of normal a- and b-cell metabolic function in
tolerant nonhuman primates. Enduring tolerance to a- and
b-cells after PIT was achieved with peritransplant admin-
istration of IT and DSG, sparing the animals from any
chronic exposure of diabetogenic immunosuppressive
drugs. These preclinical studies suggest that PIT in com-
bination with tolerance induction has exciting potential for
type I IDDM treatment.
0 10 20 30 40 50 60 70 80 90 100
20
40
60
80
100
120
140
160
180
200
220
240
Minutes
NormalIslet TransplantationSTZ treatment withoutIslet Transplantation
0 10 20 30 40 50 60 70 80 90 100
10
20
30
40
50
60
70
80
90
100
Minutes
NormalIslet TransplantationSTZ treatment withoutIslet Transplantation
A
B
*
*
Arginine
Blo
od G
luco
se (
mg/
dL)
Glu
cago
n (p
g/m
L)
Figure 6: Stepped hypoglycemic, hyperinsulinemia clamp in
STZ-induced diabetic primates, long-term survivors 2 years
after PIT, and normal controls from our colony. Stepped
hypoglycemia clamp was performed as described in Methods.
Only long-term survivors (3/7) in the group that received the
complete tolerance protocol (ITþDSG) were included. *p<0.001
vs. STZ-induced animals without PIT.
Contreras et al.
136 American Journal of Transplantation 2003; 3: 128--138
Page 10
Acknowledgments
We thank Mr Nat Borden and Martha Wilkins for outstanding care and
management of the monkeys. We appreciate Ms. Sharon McKnight’s
skilled surgical assistance.
This work is supported by NIH grant #U19-DK57958, Novartis CRADA
#639616 and JDF grant # 198242.
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