Interleukin-15 Receptor Blockade in Non-Human Primate Kidney
Transplantation1
Silke Haustein1, Jean Kwun1,*, John Fechner1, Ayhan Kayaoglu1,§, Jean-Pierre Faure1,¶,
Drew Roenneburg1, Jose Torrealba2, and Stuart J. Knechtle1,*,†.
This study was supported by: Roche Pharmaceuticals, Palo Alto, CA.
Abstract: 249 words; Text: 3543 words; References: 29
Figures: 4; Tables: 2
Key Words: Interleukin-15, Non-human primate, Transplantation, NK cells, Memory T
cells
*Present Address: Emory Transplant Center, Department of Surgery, Emory University
School of Medicine, Atlanta, GA 30322
§
Present Adress: Department of General Surgery, Gaziosmanpasa University School of
Medicine, Tokat, Turkey
¶
Present Adress: Faculté de Médecine et de Pharmacie, Université de Poitiers, 6 rue de la
Miletrie, BP 199, 86034 Poitiers cedex, France
†Address all correspondence and requests for reprints to: Stuart J Knechtle, MD,
Emory Transplant Center, 101 Woodruff Circle, 5115 WMB, Atlanta, GA 30322, U.S.A.
Phone: 404-727-7833; Fax: 404-727-3660; E-mail: stuart.knechtle@emoryhealthcare.org
Footnotes
Footnotes to The Title:
1. S.H. designed and performed research, analyzed data and wrote the paper; J.K.
participated in research design and performed research. J.F. designed research and
analyzed data; A.K. and J.P.F. participated in research (Surgery); D.R. and J.T.
analyzed data; S.J.K. participated in research design, the performance of the
research and helped to write the paper.
Footnotes to The Authors
1. Division of Transplantation, Department of Surgery, Clinical Science Center,
University of Wisconsin School of Medicine and Public Health, Madison,
Wisconsin
2. Department of Pathology and Laboratory Medicine, University of Wisconsin
School of Medicine and Public Health, Madison, Wisconsin
2
Abbreviations
ADCC, antibody-dependent cell-mediated cytotoxicity
APC, allophycocyanin
ATG, antithymocyte globulin
BUN, blood urea nitrogen
CAN, chronic allograft nephropathy
CDC, complement-dependent cytotoxicity
FCS, fetal calf serum
FITC, fluorescein isothiocyanate
H&E, hematoxylin and eosin
IL, interleukin
MHC, major histocompatibility complex
MMF, mycophenolate mofetil
PE, phycoerythrin
PerCP, peridinin chlorophyll protein
STAT, signal transducer and activator of transcription
3
Abstract
Background: Interleukin-15 (IL-15) is a chemotactic factor to T cells. It induces
proliferation and promotes survival of activated T cells. IL-15 receptor blockade in
mouse cardiac and islet allotransplant models has led to long-term engraftment and a
regulatory T cell environment. This study investigated the efficacy of IL-15 receptor
blockade using MutIL-15-Fc in an outbred non-human primate model of renal
allotransplantation.
Methods: Male cynomolgus macaque donor-recipient pairs were selected based on ABO
typing, MHC Class I typing and CFSE-based mixed lymphocyte responses. Once animals
were assigned to one of 6 treatment groups; they underwent renal transplantation and
bilateral native nephrectomy. Serum creatinine was monitored twice weekly and as
indicated; and protocol biopsies were performed. Rejection was defined as a rise in serum
creatinine to 1.5mg/dL or higher, and was confirmed histologically. Complete blood
counts and flow cytometric analyses were performed periodically post transplant; and
pharmacokinetic parameters of MutIL-15-Fc were assessed.
Results: Compared to control animals, MutIL-15-Fc treated animals did not demonstrate
increased graft survival despite adequate serum levels of MutIL-15-Fc. Flow cytometric
analysis of white blood cell subgroups demonstrated a decrease in CD8+ T cell and
natural killer (NK) cell numbers, although this did not reach statistical significance.
Interestingly, two animals receiving MutIL-15-Fc developed infectious complications,
but no infection was seen in control animals. Renal pathology varied widely.
Conclusions: Peri-transplant IL-15 receptor blockade does not prolong allograft survival
4
in non-human primate renal transplantation; however, it reduces the number of CD8+ T
cells and NK cells in the peripheral blood.
5
Introduction
Interleukin-15 (IL-15), a member of the four alpha-helix bundle family of
cytokines, has been implicated in the pathophysiology of allograft rejection (1-3). It is
produced mainly by macrophages, epithelial cells, and endothelial cells (4-7). IL-15
serves as a survival factor for T-cells; and it induces cytotoxic activity in both T cell and
natural killer (NK) cell populations (8-12). Importantly, IL-15 provides a critical survival
signal to effector memory T cells, which by virtue of their resistance to other induction
agents continue to pose a hurdle to allograft acceptance (3, 13, 14). The redundancy IL15 has with IL-2 as a survival factor, and its distinctive function in memory T cells,
therefore makes it an attractive target for novel immunosuppressive therapy.
In fact, transplant studies performed in rodents showed that blocking IL-15
signaling by blunting signaling via use of a fusion protein had a favorable effect on graft
outcomes (15). More particularly, a fusion protein was produced, in which two human
IL-15 molecules were linked to a human immunoglobulin IgG1 Fc portion with the intent
to increase circulating half-life and provide effector (i.e. CDC and ADCC) functions.
This molecule was tested in a murine model and shown to blunt delayed-type
hypersensitivity and to prolong islet allograft survival (16, 17). A regimen combining the
antagonist mutant IL-15/Fc (referred to hereafter as MutIL15-Fc) with rapamycin and an
agonist IL-2 Fc was shown to promote tolerance to vascularized cardiac grafts in rodents
(18).
On the basis of this promising data, we decided to assess the effects of MutIL15Fc in a life-supporting kidney transplantation model developed in nonhuman primates.
Our initial hypothesis was that interrupting IL-15 signaling using MutIL15-Fc would
6
result in prolongation of renal allograft survival when compared to controls treated with
subtherapeutic mycophenolate mofetil. The primary outcome measures were animal and
allograft survival, and allograft function. Secondary outcome measures were allograft
histopathology, immune cell monitoring by flow cytometry, and pharmacokinetic
monitoring of MutIL-15-Fc drug levels.
The studies detailed hereafter led to the conclusion that the molecule, while
demonstrating optimal properties in a murine system, did not perform adequately in
nonhuman primates and hence had no effect on organ allograft survival.
7
Materials and Methods
Animals
Five- to six-year old male cynomolgus macaques of Chinese and Vietnamese origin,
weighing 7.6±0.7 kg, underwent ABO typing, MHC Class I typing by reference strand
mediated conformational analysis (19), and blood draws for assessment of
carboxyfluorescein succinimidyl ester-based mixed lymphocyte response. Donorrecipient pairs were selected based on results from these tests. Animals were assigned to
one of 6 treatment groups (Table 1). Some animals served as a donor twice, others were
recipients who underwent bilateral native nephrectomy at the time of transplantation.
When possible, animals first served as a donor, and after a one month period of recovery,
underwent renal allotransplantation with completion native nephrectomy. Two animals
underwent renal autotransplantation of the left kidney, keeping the right native kidney in
place. All surgical procedures were performed under sterile conditions. Animals were
housed in accordance National Institutes of Health and U.S. Department of Agriculture
guidelines; and all animal research was approved by the University of Wisconsin
Institutional Animal Care and Use Committee.
Study Drug
IL-15 receptor blockade was achieved using a protein, MutIL-15-Fc, that consists
of two mutated IL-15 molecules attached to a humanized IgG1 Fc chain and that was
provided by Roche (Firma Hoffmann-La Roche, Palo Alto, CA) (Figure 1a). The
molecule, originally developed in murine systems, was shown to selectively bind to IL-
8
15R with high affinity (17). It is devoid of agonistic properties and does not interfere
with IL-2 and IL-3 signaling (17). The molecule used in this study was produced and
expanded at Roche and shown to have similar biochemical characteristic as the original
molecule (data not shown). The effector function afforded by the Fc portion of the
murine version of the molecule was shown to be essential to its activity as mutations
introduced in the region abolished its activity (15, 20).
Treatment Groups
A total of 11 animals were recipients of renal transplants. Animals were assigned
to one of six treatment groups, as outlined in Table 1. Low-dose MutIL-15-Fc was
administered as an intraoperative 2mg/kg intravenous infusion just prior to reperfusion;
and then subcutaneously twice per week starting on postoperative day one. High-dose
MutIL-15-Fc was administered by daily subcutaneous injections, beginning one week
prior to transplantation, and continuing until animal sacrifice. MMF was administered as
an oral solution, beginning at 20mg/kg twice daily, and subsequently titrating the dose to
a target trough level of 2ng/mL. MMF levels were measured twice per week. ATG
therapy started on day -1 (day 0 being the day of transplant) with a 5mg/kg intramuscular
injection; on day 0 ATG was given at 5mg/kg intravenously just prior to reperfusion; and
on days 1, 3, and 5 ATG was given at 5mg/kg subcutaneously. ATG was given after pretreatment with diphenhydramine and/or methylprednisolone.
Clinical and Laboratory Assessments
9
Animal and graft survival were measured from day 0, the day of
transplantation, to the day of sacrifice or death. Renal allograft function was
monitored by urine output (high, medium, low, and none) daily for 14 days and then
three times per week by inspection of the collection pan. Serum BUN and serum
creatinine were measured on postoperative days 2, 3, 5 or 6, and then twice per week.
Acute rejection was defined as three or more of the following: serum creatinine greater
than 6 mg/dl, anuria for 2 days, abnormal behavior, acute weight loss >10% during the
previous two weeks, chronic weight loss of >10% of day 0 weight, ascites or severe
edema associated with serum protein <3.5 mg/dl, kidney biopsy indicating rejection,
or proteinuria (>100 mg/dL protein).
To monitor for drug side effects, complete blood counts with differential and
serum chemistries were obtained twice per month. To monitor physical well-being,
weight was monitored every time an animal was sedated for other purposes.
Histology and Pathology
Renal tissue specimens were obtained by biopsy on postoperative days 6 or 7, and
28, and by nephrectomy at the time of native nephrectomy and necropsy. Tissues were
fixed with 10% buffered formalin and submitted for processing into paraffin blocks.
Paraffin blocks were then sectioned at 5 microns and the representative tissue sections
stained with Hematoxylin and Eosin (H&E) for microscopic evaluation. Samples were
submitted and read by a trained renal pathologist (JRT). All animals underwent a
complete necropsy by the Wisconsin National Primate Research Center’s pathologists.
10
Flow Cytometry
Peripheral blood was collected at baseline, necropsy, and at weekly intervals, in
heparin- or EDTA-coated tubes. Briefly, 100μL blood were incubated with 7μL of each
antibody for 20 minutes at 4˚C in the dark. Cells were then lysed with BD FACS Lyse
(BD Biosciences, San Jose, CA) at room temperature for 10 minutes in the dark, and
washed twice with FACS buffer (1% FCS in PBS). Prior to analysis on a FACSCalibur
flow cytometer (BD Biosciences, San Jose, CA), cells were fixed in 1%
paraformaldehyde. Data was acquired with CellQuestPro Software (BD Biosciences, San
Jose, CA), until 25,000 lymphocytes events were counted, or until 2 minutes had passed.
On the FACSCalibur, 488 excitable dyes included FITC, PE, and PerCP. The FITC
signal was collected through a 530/30 band pass filter, the PE signal was collected
through a 585/42 band pass filter, and the PerCP signal was collected with a 670 long
pass filter. The signal from APC, a 633 excitable dye, was collected with a 660/20 band
pass filter. Data was analyzed using FloJo software (Version 8.4.3, Tree Star Inc.,
Ashland, OR). Absolute cell numbers were calculated using the most recent white blood
cell count.
The following antibodies (clones) were used (BD Biosciences, San Jose, CA):
CD4 FITC (L200), CD8 FITC (RPA-T8), mIgG1κ FITC, CD8 PE (RPA-T8), CD16 PE
(3G8), CD20 PE (2H7), CD69 PE (FN50), CD95 PE (DX2), mIgG1κ PE, CD3 PerCP
(SP34.2), mIgG1λ PerCP, CD3 APC (SP34.2), CD14 APC (M5E2), CD25 APC
(MA251), CD28 APC (CD28.8), mIgG1κ APC.
Pharmacokinetics
11
Serum samples were collected immediately after intravenous infusion (IV peak
level), on the morning of postoperative day 1 (IV trough level), 13 and 24 hours after the
first subcutaneous dose (13 and 24 hour peak levels), and on the morning of
postoperative day 3 (SC trough level). Therapeutic drug levels were achieved in all
animals.
Data Analysis
Survival data are presented in a descriptive format due to low group numbers.
Differences in laboratory values from baseline to necropsy were compared using a twotailed paired t-test (GraphPad Prism Software, Version 4.00, San Diego, CA).
12
Results
Animal and graft survival, and graft function
Allograft survival times are summarized in table 2. Two animals, Cy0059 and
Cy0110, were treated with low-dose MutIL-15-Fc monotherapy, with their grafts being
lost due to acute rejection on postoperative day 6. Two animals, Cy0060 and Cy0076,
underwent allotransplantation and were treated with mycophenolate mofetil
monotherapy. One graft (Cy0060) was lost on day 2 because of anuria; which was in
retrospect attributable to hydronephrosis; the other (Cy0076) showed stable graft function
until acute rejection on day 30. Because of the poor outcome of the first two animals, we
decided to pursue two allotransplants (Cy0092 and Cy0100) at a higher dose of MutIL15-Fc. One graft (Cy0092) was lost due to renal artery thrombosis; the other was lost on
postoperative day 2 due to anuria and severe rejection. Because of these early rejection
episodes, we performed two autotransplants to examine whether it was truly rejection, or
whether other factors, such as ischemia-reperfusion injury or drug toxicity, were
contributing causes to early allograft loss. Both autotransplanted animals (Cy0056 and
Cy0062) were sacrificed as scheduled on postoperative day 14 with presumably
functioning autografts to compare the histology of their remaining native kidney to that
of the autotransplanted kidney. Since these autografts histologically recovered by day 14,
we proceeded to evaluate low-dose MutIL-15-Fc treatment in combination with ATG and
MMF. These two grafts, from Cy0052 and Cy0108, were lost on days 7 and 20,
respectively, due to renal failure (Cy0052), and due to viral hepatitis (Cy0108) with
stable graft function.
13
Treatment with MutIL-15-Fc monotherapy at either dose did not prolong renal
allograft survival when compared to subtherapeutic MMF, or when compared to
untreated historical controls, which generally experience acute rejection between days 68 (data not shown). Interestingly, both animals in the ATG/MMF/low-dose MutIL-15-Fc
group did not demonstrate any signs of rejection or other allograft injury at necropsy (d7,
d20), which compares favorably to the day 7 biopsy result of the ATG/MMF treated
animal that showed sclerosis grade I.
Laboratory test changes
In terms of complete blood count, hemoglobin and hematocrit were significantly
decreased at necropsy when compared to baseline in all animals (paired t-test, p=0.002
and 0.003), which is likely due to frequent phlebotomy and hemodilution. White blood
cell counts and platelet counts were not different between baseline and necropsy.
The following laboratory values were significantly lower at necropsy when
compared to baseline: glucose (p=0.03), total protein (p=0.002), albumin (p<0.0001),
iron (p=0.0015), sodium (p=0.0204), and chloride (p= 0.0037). LDH (p=0.04) and
phosphate (p=0.0166) were increased at necropsy. No changes were observed in
cholesterol, triglycerides, creatine kinase, alanine aspartate transferase, total bilirubin,
gamma glutamyl transferase, alkaline phosphatase, calcium, potassium, and animal
weight.
Histology and Pathology
14
Native kidneys were stained with H&E as normal controls. Mild tubular injury
was occasionally seen in native untreated kidneys; otherwise, native untreated kidneys
appeared histologically unremarkable. Animals in the high dose MutIL-15-Fc group were
treated for one week prior to transplantation and native nephrectomy. Interestingly, the
native treated kidneys of these animals showed moderate diffuse tubular injury on
histologic examination despite stable renal function.
Day 7 protocol kidney biopsies were performed in 5 animals. In the autotransplant
group, one animal showed findings of acute interstitial nephritis and acute tubular injury.
This animal had a prolonged warm ischemic time intraoperatively and was given heparin
due to recurring intraoperative renal artery thrombosis. Because multiple factors may
account for the interstitial nephritis seen in this animal, we were hesitant to draw firm
conclusions from this finding. The second autotransplanted animal had no tubular
damage, and showed only mild focal segmental glomerular hypercellularity, which can
be a normal finding in young animals. Two control animals, one on MMF monotherapy
(Cy0076) and the other on ATG/MMF (Cy0055), had day 7 biopsies. These showed a
focal lymphoid aggregate, and sclerosis grade I, respectively. Of note, only one animal
treated with MutIL-15-Fc, ATG, and MMF who underwent allotransplantation survived
to a day 7 biopsy (Cy0108); it died on day 20 from viral hepatitis with stable graft
function. This day 7 renal biopsy was suspicious for acute rejection and showed mild
tubular injury (Figure 2).
Renal pathology findings at necropsy are listed in Table 2. Acute rejection with
diffuse interstitial hemorrhage was seen in the necropsy specimen of both animals treated
with low-dose MutIL-15-Fc. In the renal autotransplant group, one animal showed a
15
normal appearing autograft, while the animal that had a prolonged warm ischemic time
had resolving tubulointerstitial nephritis. Both animals treated with high-dose MutIL-15Fc had diffuse tubular injury, but no rejection on day 1 and 2 after transplant,
respectively, although one was lost due to arterial thrombosis. The MMF and ATG/MMF
control animals all showed signs of acute rejection at necropsy. Lastly, animals treated
with ATG/MMF/low-dose MutIL-15-Fc did not show signs of rejection in their
transplant kidneys at necropsy, because they were lost earlier for other reasons. If they
had survived long-term, these animals would likely still have developed acute rejection at
a later timepoint.
Flow Cytometry
In all animals, whether controls or treated animals, a decrease in absolute CD8+
and CD4+ T cell numbers was always seen in peripheral blood after transplantation. The
two animals treated with high dose MutIL-15-Fc are of particular interest here; because
they were treated with study drug for one week prior to transplantation. Therefore, they
have data at baseline (untreated), pre-transplant (after one week of treatment with 6mg/kg
SC daily), and post-transplant. Figure 3 shows the CD8+ T cell, CD4+ T cell, and NK cell
responses to treatment, and to transplant, in these animals. CD8+ T cells, as well as NK
cells declined in absolute number with drug treatment, whereas CD4+ T cells remained
stable or experienced a mild increase in absolute numbers.
CD8+ T cell subsets were categorized into three groups according to Pitcher’s
description (24): CD28+CD95high cells were designated as “central memory” CD8+ T
cells; CD28+CD95low cells were designated as “naïve” CD8+ T cells, and CD28-
16
CD95intermediate cells were designated “effector memory” CD8+ T cells (Figure 4A). Based
on these designations, the following observations were made: All three CD8+ T cell
subsets declined in all animals after transplantation, including MMF alone and
ATG/MMF treated control animals (data not shown). Again, evaluating the two animals
that received high-dose MutIL-15-Fc treatment for one week prior to transplantation, a
decrease in naïve, central, and effector memory CD8+ T cells was seen prior to
transplantation, which may be attributable to drug effect, or to normal day-to-day
variation in white blood cell subsets. After transplantation, a further decline in overall
CD8+ T cells, and in central and effector memory CD8+ T cell subgroups was seen. The
changes in naïve CD8+ T cells are less clear after transplantation, as one animal had an
increase and the other a decrease in absolute naïve CD8+ T cell numbers (Figure 4B).
17
Discussion
Interruption of the IL-15 signaling pathway has in the past shown significant
effects in rodent models. Smith et al (21) demonstrated prevention of rejection of minor
MHC mismatched cardiac allografts in mice and a prolongation in survival of major
MHC mismatched cardiac allografts in combination with non-depleting anti-CD4
antibody, using a soluble IL-15R molecule. Subsequently, Lacraz et al demonstrated
that pancreatic islet allografts were permanently accepted in partially MHC mismatched
mice treated with a combination of IL-15 mutant/Fc2a and CTLA4/Fc treatment (16, 22,
23). Zheng et al published their experience with a mutant IL-15/Fc protein. In their model
of minor MHC mismatched mouse cardiac allotransplantation, treatment with mutant IL15/Fc protein induced permanent cardiac allograft survival. Furthermore, they delineated
differences in the dosing of mutant IL-15/Fc and the effects of dosing on cardiac allograft
survival (15).
When using MutIL-15-Fc in a monkey model of renal allotransplantation, animal
and allograft survival were not prolonged. Single drug therapy did not prevent acute
rejection within one week of transplantation; using MutIL-15-Fc in combination with
ATG and MMF also did not prolong allograft survival when compared to controls.
However, in both animals treated with triple combination therapy, no rejection or other
allograft injury was seen at necropsy. It is unfortunate that these animals were lost for
reasons other than acute rejection. The fact that one succumbed to a viral infection may
be indicative of over-immunosuppression. However, it remains unclear whether this is
attributable to an additional immunosuppressive effect of MutIL-15-Fc over ATG/MMF
therapy alone.
18
The histopathologic findings at necropsy raise some interesting questions in
regard to the role of IL-15. Low-dose MutIL-15-Fc therapy resulted in acute rejection IB,
with severe tubular injury, diffuse interstitial nephritis, and fibrinoid necrosis with
hemorrhage at necropsy, consistent with acute rejection. From the literature, we also
know that IL-15 is constitutively expressed by human renal tubular epithelial cells; and
that IL-15 production increases in the presence of interferon-γ (7, 24). Prior studies
interfering with IL-15 signaling in mice had only addressed islet and cardiac, but not
renal transplantation; and renal failure was not reported in these models (16, 21-23).
However, Shinozaki et al showed that IL-15 protects tubular epithelial cells from
undergoing apoptosis in a mouse model of nephrotoxic serum nephritis (25). Therefore,
we were concerned that MutIL-15-Fc treatment might predispose tubular epithelial cells
to apoptosis, possibly contributing to the tubular injury seen. Consequently, we chose to
begin treatment of the high-dose MutIL-15-Fc treated animals one week prior to
transplantation, to give us a chance to evaluate renal function clinically and histologically
in the absence of ischemia-reperfusion and alloimmune injury. After one week of highdose MutIL-15-Fc therapy, moderate diffuse tubular injury to the native kidneys was seen
at the time of transplantation despite clinically stable renal function.
The question remained whether IL-15 provides an essential survival signal to
tubular epithelial cells survival after ischemia/reperfusion or alloimmune injury. The
findings of our autotransplants demonstrate that IL-15 blockade in the presence of
ischemia-reperfusion injury (without alloimmune injury) is not detrimental to renal
tubular epithelial cells. The interstitial nephritis seen in one animal has multiple possible
causes, including prolonged warm ischemic time, administration of heparin, an
19
idiosyncratic reaction to prophylactic antibiotics or other drugs, or even a viral etiology.
Of note is the fact that naïve animals chronically exposed to the compound for a month
did not show indication of kidney damage (data not shown).
A decrease in all lymphocyte cell numbers was consistently seen after renal
transplantation in all animals, whether treated with or without study drug. This made it
difficult to discern any effects of MutIL-15-Fc. The most interesting treatment group,
therefore, consisted of the high-dose MutIL-15-Fc treated animals, which were treated for
one week prior to transplantation with MutIL-15-Fc. A decrease in CD8+ T cell and NK
cell numbers was seen during the week prior to transplantation, as was a decrease in
CD8+ naïve, central memory, and effector memory T cell subsets. One exciting finding is
that treatment with MutIL-15-Fc appears to reduce the CD8+ effector memory T cell
subpopulation more than other CD8+ T cell subsets, although we could not confirm this
statistically due to low animal numbers. This effector memory T cell pool has been noted
to be resistant to depletion by other induction agents (26, 27). Indeed, elevated IL-15
levels have been documented in chronic allograft rejection (1-3, 28). In addition to
serving as a survival factor for T-cells, and inducing cytotoxic activity in T and NK cells,
IL-15 boosts the development and preferential survival of CD8+ memory T cells (13, 14),
which are implicated in chronic rejection and remain a hindrance to the development of
permanent allograft acceptance (1-3). Because these decreases in absolute lymphocyte
cell numbers were a consistent finding in both animals, we believe that MutIL-15-Fc
indeed has the effect of decreasing CD8+ T cell and NK cell numbers, as well as certain
CD8+ T cell subsets. Still, this finding is based on data from only two animals, and while
very exciting, it is to be interpreted with caution for now.
20
The pharmacokinetic parameters of MutIL-15-Fc correlated closely with those
predicted by pharmacokinetic modeling. All animals showed therapeutic levels of MutIL15-Fc, thus making it unlikely that rejection was due to poor drug exposure.
In conclusion, our results suggest that MutIL-15-Fc treatment alone is not
sufficient to prevent allograft rejection in a monkey model of renal allotransplantation.
The effects of IL-15 blockade on CD8+ memory T cells and on NK cells are novel and
exciting, but must be proven true and effective in future experiments. MutIL-15-Fc is a
reagent capable of blocking cytokine signaling in rodents, but does not perform
identically in primates. Such inconsistencies between species are not unheard of; in fact,
some species specificities were reported for the IL-2 receptor as well (29). The
mechanisms underlying allograft rejection and allograft acceptance remain incompletely
understood and deserve further investigation in animal models.
21
Acknowledgements
We thank Kathy Schell and Joel Puchalski from UWCCC for assistance with flow
cytometry and the WNPRC staffs for animal care work. We also thank Amanda
Kolterman and Dr. Julio Pascual for help in conducting the experiments and for critical
reviews of this manuscript.
22
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Figure Legends
Figure 1. Measured graft function by serum creatinine and BUN from low dose mutiL15-Fc treated allotransplant (A), low dose mut-iL15-Fc treated autotransplant (B), High
dose mut-IL-15-Fc treated allotransplant (C), MMF treated allotransplant (D),
ATG/MMF treated allotransplant (E), and ATG/MMF/low dose mut-IL-15-Fc treated
allotransplant. Graft function was measured twice per week by serum creatinine (solid
line) and serum BUN (interrupted line). Data are presented by treatment group for each
animal. Both autotransplanted animals in (B) were sacrificed with normal renal function,
but both still had a native kidney in place. The only other animal that had normal renal
function at death was Cy0108 in (F). This animal died from viral hepatitis.
Figure 2. Histopathology of non-human primate renal biopsy. Representative
histopathologic results are given for the four treatment groups that included mut-IL-15-Fc
as part of the immunosuppressive regimen. In animals treated with low-dose mut-IL-15Fc, native kidneys had a normal appearance (A). At necropsy, acute rejection, diffuse
interstitial nephritis, diffuse interstitial hemorrhage, and severe tubular injury were seen
(B). In animals treated with high-dose mut-IL-15-Fc, native kidneys were treated for one
week prior to native nephrectomy. These native, treated kidneys show moderate diffuse
tubular injury; however, clinically, kidney function was preserved (C). At necropsy,
severe diffuse tubular injury is seen (D). Autotransplanted animals continued to have the
right native kidney (E) in place while the left kidney was transplanted (F) into the lower
abdomen. Both kidneys were treated for 14 days with mut-IL-15-Fc; and both kidneys
28
appeared normal at necropsy. Two animals were treated with ATG/MMF and low-dose
mut-IL-15-Fc. Both showed no acute rejection; although the day 7 biopsy for Cy0108
was “suspicious” (G). At necropsy, there was no acute rejection; only a minimal focal
mononuclear interstitial infiltrate was seen (H).
Figure 3. Response of T and NK cells to mut-IL-15-Fc treatment. Number of CD4 T cell,
CD8 T cell and NK cell were analyzed with flow cytometry. Decrease in NK and CD8 T
cells of two animals while on treatment with high-dose mut-IL-15-Fc pre-transplant and
again post-transplant as treatment continued. No such decrease was seen in and CD4 T
cells pre-transplant.
Figure 4. Flow Cytometric Delineation of CD8 T Cell Subsets. (A) Gating strategy for
memory/naïve T cell population demonstrates CD8 T cell subsets pre-transplant: Naïve
cells are CD28+CD95low; central memory T cells are CD28+CD95high; and effector
memory T cells are CD28-CD95intermediate. (B) Effector memory T cells decrease while on
treatment with mut-IL-15-Fc pre-transplant, and continue to decrease post-transplant.
Central memory T cells also show a slight decrease pre- and post-transplant. Naïve T
cells decrease mildly pre- and post-transplant in Cy0092; whereas they first decrease and
then increase in Cy0100.
29
Table 1. Treatment Groups
Group
Na
Mut-IL-15-Fc Dose
MMF dose
ATG dose
n/a
n/a
n/a
n/a
n/a
n/a
2mg/kg IVb d0
Low-dose Mut-IL-15-Fc (allotransplant)
2
1mg/kg SCc twice weekly
2mg/kg IVb d0
Low-dose Mut-IL-15-Fc (autotransplant)
2
1mg/kg SCc twice weekly
6mg/kg SCc daily starting 1 week
High-dose Mut-IL-15-Fc (allotransplant)
2
pre-transplant
20mg/kg POe BIDf
MMF alone (allotransplant)
2
n/a
n/a
then titrated
5mg/kg IMd d -1
e
20mg/kg PO BID
ATG/MMF (allotransplant)
1
f
5mg/kg IVb d0
n/a
then titrated
5mg/kg SCc d1, 3, and 5
5mg/kg IMd d -1
2mg/kg IVb d0
ATG/MMF/low-dose Mut-IL-15-Fc
2
(allotransplant)
a
c
1mg/kg SC twice weekly
20mg/kg POe BIDf
5mg/kg IVb d0
then titrated
N, number; bIV, intravenous; cSC, subcutaneous; dIM, intramuscular; ePO, orally; fBID, twice daily
5mg/kg SCc d1, 3, and 5
Table 2. Summary of Allograft Survival and Pathologic Findings
Group
n
ID
Graft Survival
(days)
Low-dose (allotransplant)
Low-dose (autotransplant)
High-dose (allotransplant)
MMF alone (allotransplant)
2
2
2
2
Renal Pathology at
Diagnoses at Necropsy and Comments
Necropsy
Cy0059
6
ARa IB, severe TIb
acute glomerulitis and diffuse interstitial
Cy0110
6
ARa IB, severe TIb
nephritis in both
Cy0056
14
TINc
Prolonged WITd in Cy0056
Cy0062
14
Normal
Cy0092
1
No ARa, moderate TIb
Cy0100
2
No ARa, severe TIb
Cy0060
2
ARa IA, TIb
Cy0076
30
ARa IIA
Renal artery thrombosis in Cy0092
Hydronephrosis in Cy0060
ATG/MMF (allotransplant)
1
Cy0055
43
ARa IIB
ATG/MMF/low-dose (allotransplant)
2
Cy0052
7
No ARa
Partial hydronephrosis in Cy0052
Cy0108
20
No ARa
Viral hepatitis in Cy0108
a
AR, acute rejection; bTI, tubular injury; cTIN, tubulointerstitial nephritis; dWIT, warm ischemic time