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Department of Medicine, Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2a
protein, a potentially cytolytic protein that is also a high-affinity
receptor site specific antagonist for the IL-15R
receptor protein,
as a therapeutic agent. The IL-15-related fusion protein was used as
monotherapy or in combination with CTLA4/Fc in murine islet allograft
models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fc2a were
comparably effective in a semiallogeneic model system, and combined
treatment with IL-15 mutant/Fc2a plus CTLA4/Fc produced universal
permanent engraftment. In a fully MHC-mismatched strain combination
known to be refractory to costimulation blockade treatment, combined
treatment with both fusion proteins proved to be highly effective;
>70% of recipients were tolerized. The analysis revealed that the
IL-15 mutant/Fc treatment confers partial protection from both
CD4+ and CD8+ T cell graft infiltration. In
rejections occurring despite CTLA4/Fc treatment, concomitant treatment
with the IL-15 mutant/Fc2a protein blocked a CD8+ T
cell-dominated rejection processes. This protection was linked to a
blunted proliferative response of alloreactive T cells as well
silencing of CTL-related gene expression events. Hence, we have
demonstrated that targeting the IL-15/IL-15R pathway represents a new
and potent strategy to prevent costimulation blockade-resistant
CD8+ T cell-driven rejection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clearly, costimulation blockade drastically decreases expression of
IL-2 posttransplantation (7, 9). Which T cell growth
factors are responsible for the clonal proliferation of alloreactive T
cells that cause graft rejection in some costimulation blockade-treated
hosts? IL-2 expression is not a prerequisite for allograft rejection as
IL-2 knockout (KO)5
mice promptly reject islet and cardiac allografts (16, 17). Immunohistology in the IL-2 KO recipients reveals that
CD4+ and CD8+ T cells
infiltrate rejecting grafts while robust intragraft expression of the
IL-4, IL-7, IL-15, as well as granzyme B genes is evident (Refs.
16, 18 and our unpublished data). Moreover, mice rendered
genetically deficient for both IL-2 and IL-4, the principal T cell
growth factors that are produced by T cells, also readily reject
allografts (17). Thus, non-T cell-derived cytokines, such
as IL-15, may be actively involved in supporting allograft rejection.
An analysis of biopsies taken from rejecting human renal allografts has
revealed that intragraft gene expression of IL-15 is a far
more consistent marker of rejection than IL-2 gene
expression (18). Moreover, Smith et al. (19)
have recently reported the prolonged survival of heart allografts in a
treatment protocol consisting of soluble IL-15R chain proteins and
nondepleting anti-CD4 mAb. Can expression of IL-15 be linked to
CD8-dependent costimulation-resistant rejection? The phenotype of
IL-15-/- (20) and
IL-15R-/- (21) mice
demonstrates that IL-15 selectively supports the homeostasis and
proliferation of memory CD8+ T cells (22, 23).
Costimulation blockade cannot act to directly decrease IL-15 expression because epithelial and endothelial cells and monocyte/macrophages, not T cells, are the primary cellular sources of this cytokine (24). Since costimulation blockade-resistant rejection may be mediated by CD8+ T cells and IL-15 is essential for the homeostasis and proliferation of CD8+ T cells, we have now tested the hypothesis that targeting the IL-15/IL-15R system in concert with costimulation blockade might provide a new tool for the induction of allograft tolerance.
The trimolecular IL-15R expressed upon T cells includes the
constitutively expressed IL-2R
and IL-2R common -chains shared
with the IL-2R. A unique IL-15R chain (25, 26) is
expressed upon activated, but not resting, mononuclear leukocytes
(27). The IL-15R is expressed in lesser abundance upon
activated epithelial cells, macrophages, and fibroblasts
(24). We have previously reported on the
specificity of a unique IL-15 mutant/Fc2a protein possessing a
prolonged circulating half-life, high-affinity IL-15R site-specific
antagonist function and both complement- and Fc-related cytocidal
potential against IL-15R+ cells
(28). Kim et al. (28) have demonstrated that
this IL-15 mutant/Fc fusion protein specifically binds to the
IL-15R; IL-15 mutant/Fc2a binding to activated T cells was
blocked by IL-15, but not by IL-2 or anti-IL-2/IL-15R chain Abs.
In this report, we also demonstrated that this IL-15 mutant/Fc protein
efficiently blocks delayed-type hypersensitivity in mice
(28). In the present report, the therapeutic activity of
the IL-15-related protein was compared with that of a CTLA4/Fc.
CTLA4/Fc treatment is known to promote engraftment and often leads to
tolerance (5, 29). Combined treatment with CTLA4/Fc and
IL-15 mutant/Fc proteins was also studied. Our data suggest that
IL-15R+ cells are an important component of
allograft rejection and these cells play a pivotal role in
CD8+ T cell-mediated CTLA4/Fc blockade-resistant
rejection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c (H-2d), DBA/2 (H-2d), B6AF1 (H-2b/d.k), and C57BL/6 (H-2b) mice, 8–10 wk old, were obtained from Taconic Farms (Germantown, NY). C57BL/6cd4tm1knw (CD4KO, H-2b) and C57BL/6.129S2cd8atm1mak (CD8KO, H-2b) were obtained from The Jackson Laboratory (Bar Harbor, ME).
Islet transplantation
Allogeneic DBA/2 islet cell grafts were transplanted into 8- to
10-wk-old B6AF1 recipient mice rendered diabetic
by a single i.p. injection of streptozotocin (225 mg/kg; Sigma, St
Louis, MO). Allogeneic BALB/c islet cell grafts were transplanted into
8- to 10-wk-old C57BL/6 recipient mice rendered diabetic by a single
i.p. injection of streptozotocin (270 mg/kg). Islet cell
transplantation was performed as previously described
(30). Briefly, islets were isolated from donor DBA/2 or
BALB/c pancreata through collagenase digestion and centrifugation on a
discontinuous Ficoll gradient. The crude islet isolates containing
islets, vascular tissue, ductal fragments, and lymph nodes were divided
into aliquots of 
300 islets and were transplanted under the renal
capsule into B6AF1 or C57BL/6 recipients. By
intent, we use these crude islet preparations which are more
immunogenic than more pure islet preparations (our unpublished
observations). Initial allograft function was verified by sequential
blood glucose measurements with levels under 200 mg/dl on days 3–5
after transplantation, and graft rejection was defined as a rise in
blood glucose levels exceeding 300 mg/dl following a period of primary
graft function.
Treatment protocol
Murine CTLA4/Fc (31) and human IL-15 mutant/Fc2a
(28) proteins were constructed and expressed in our
laboratory. CTLA4/Fc protein used in these studies bears active FcR
binding and complement-binding domains (29). Treatment of
islet allograft recipients with CTLA4/Fc consisted of 0.1 mg/day i.p.
for 10 consecutive days after transplantation, which is the optimal
treatment period in this model (our unpublished data). Islet allograft
recipients received 1.5 µg of IL-15 mutant/Fc2a/day i.p. for 21
consecutive days after transplantation. An IgG2a protein, bearing the
same Fc sequence as the CTLA4/Fc and IL-15 mutant/Fc2a proteins, was
used as a control treatment at 1.5 µg/day for 10 days.
Histopathology and immunohistology
The left kidney bearing the islet graft was removed from the recipients after 8 days and embedded in OCT compound (Tissue TCK; Miles Scientific, Elkhart, IN). Cryostat sections of islets (n = 3/group) were fixed in paraformaldehyde-lysine-periodate for analysis of leukocyte Ags and stained by a four-layer peroxidase-antiperoxidase method involving overnight incubation with mAb, followed by mouse Ig-absorbed goat anti-rat Ig, rabbit anti-goat Ig, goat peroxidase-antiperoxidase complexes, and diaminobenzidine substrate. Rat anti-mouse mAbs and isotype-matched control mAbs were purchased from BD PharMingen (San Francisco, CA) and included mAbs to CD4+ (H129.19) and CD8+ (53-6.7) T cells. Sections were counterstained in hematoxylin and mounted. Isotype-matched mAbs and a control Ab were analyzed for endogenous peroxidase activity in each experiment. Samples were assigned a random number and processed and evaluated in a blinded fashion; each sample was evaluated at two to three different levels of sectioning.
PCR analysis
Intragraft mRNA analysis was performed via template RT-PCR as
previously described (16). The specific primers used for
hybridization to murine IL-2, TCR C, perforin, Fas ligand (FasL),
granzyme B, and GAPDH cDNA, the latter as an internal control, have
been previously described (16). The PCR amplification was
performed in a thermocycler (Gene Amp, PCR system 2400;
PerkinElmer/Cetus, Norwalk, CT): denaturing at 94°C for 30 s,
annealing at 57°C for IL-2 and FasL, at 60°C for perforin and
granzyme B, at 60°C for TCR C, and at 57°C for GAPDH for 30
s, and extension at 72°C for 30 s for each cycle, for a total of
40 cycles. A negative control was included for each PCR amplification
and consisted of the omission of cDNA in the PCR mixture. To further
confirm data, a semiquantitative competitor template RT-PCR analysis
was also performed with IL-2 using the gene-specific relative RT-PCR
kit from Ambion (Austin, TX). To quantitate the relative amounts of TCR
C gene transcripts between samples, each PCR was performed by
coamplifying the cDNA of interest with an internal PCR control. A
gene-specific competitive template cDNA was designed and the
semiquantitative PCR was performed as described by Steiger et al.
(16). After amplification, the samples (10 µl) were
separated on ethidium bromide-stained 1.5% agarose gel and the DNA
were visualized and photographed using UV transilluminator (Gel Doc
1000; Bio-Rad, Hercules, CA).
CFSE labeling and analysis of T cell proliferation in vivo
Spleen and lymph node cells from wild-type C57BL/6,
CD8-/-, or CD4-/- mice
(C57BL/6, H-2b) were harvested, processed, and
labeled with CFSE (Molecular Probes, Portland, OR) as described
previously (32). CFSE was dissolved in DMSO and added into
the cell suspension at a final concentration of 5 nM for 3 min at room
temperature. The reaction was stopped by the addition of HBSS/1% FCS.
The cells were washed in HBSS/1% FCS and resuspended in the same
solution before injection. Recipient BALB/c mice were sublethally
irradiated (1000 rad with a GammaCell irradiator, Kanata, Ontario,
Canada) before injection of the CFSE-labeled cells via the
lateral tail vein. From that day, untreated recipient mice or mice
receiving an i.p. injection of CTLA-4/Fc (0.1 mg/mouse) or IL-15
mutant/Fc2a protein (1.5 µg/mouse) for 3 days were studied.
Adoptive transfer experiments using syngeneic CFSE-labeled lymphocytes
was also performed as a control. On day 3, recipient spleen and lymph
node cells were removed and cell suspensions were processed as before.
Cells were stained with anti-CD4-PE conjugate (L3T4, 2 µg/ml) or
a biotinylated mAb against mouse CD8a (53-6.7, 2 µg/ml; BD
PharMingen) for 30 min at 4°C. After staining, cells were washed once
and resuspended in 0.5 ml of HBSS for analysis by flow cytometry using
a BD Biosciences FACSort equipped with CellQuest software (Mountain
View, CA). Live events were collected and analyzed by gating on
CD4+ or
CD8+CFSE+ cells.
Calculation of the frequency of proliferating T cell
Analysis of CD4+ and CD8+ T cell proliferation in response to alloantigen stimulation was performed according to Noorchashm et al. (33). With each round of cell division, the CFSE dye partitions equally between the daughter cells. By using the FACS acquisition software (CellQuest), the total number of cells in each generation of proliferation can be calculated and the number of precursors that generated the daughter cells was determined by using the following formula: y/n2 (y = absolute number of cells in each peak, n = number of cell division). The calculation of the frequency of T cell proliferation was then analyzed by dividing the total number of precursors by the total CFSE-labeled cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To probe for the role of the IL-15/IL-15R network in the allograft
response, partially MHC-mismatched DBA/2 (H-2d)
islet allografts were transplanted into B6AF1
(H-2b/d.k) mice. Untreated recipients rejected
DBA/2 islet allografts with a mean survival time (MST) of 14 days
(Table I
) while
B6AF1 recipient mice treated with CTLA4/Fc or
IL-15 mutant/Fc2a have a MST of 70 and 77 days, respectively. All
B6AF1 recipients of DBA/2 islet allografts
treated with a combination of IL-15 mutant/Fc2a and CTLA4/Fc were
permanently engrafted. Surgical removal of the left kidney bearing the
islet allograft was performed on two recipients 150 days after
transplantation. Six days later, a second DBA/2 islet allograft was
successfully engrafted without rejection in the absence of further
immunosuppressive therapy.
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a). As additional controls,
C57BL/6 recipients were also treated with a control IgG2a protein (1.5
µg/ml). In other experiments, C57BL/6 received CTLA4/Fc (0.1 mg/ml)
plus IgG2a (1.5 µg/ml). Recipients treated with IgG2a rejected
rapidly their allografts (MST, 14 days; Fig. 1a), and the
recipients treated with CTLA4/Fc and IgG2a rejected allografts at the
same tempo as recipients treated with CTLA4/Fc alone (Fig. 1a) These data confirm that the prolonged engraftment
observed with IL-15 mutant/Fc2a fusion protein treatment is not
produced solely by Fc sequences shared with IgG2a. Engraftment was
prolonged in C57BL/6 recipients treated with CTLA4/Fc or with IL-15
mutant/Fc2a protein (MST, 30 days; Fig. 1a); some grafts
functioned permanently following monotherapy with either agent. A
dramatic improvement was noted among C57BL/6 recipients treated with a
combination of IL-15 mutant/Fc2a and CTLA4/Fc (MST 120 days; Fig. 1a). Surgical removal of the islet allograft in each of five
recipients 150 days after transplantation led to hyperglycemia,
demonstrating that the euglycemic state was maintained by the islet
allografts. Subsequent to removal of the primary allograft, four
recipient mice were successfully engrafted with a second BALB/c islet
allograft in the absence of further immunosuppressive treatment (Fig. 1b). One recipient received a third-party islet allograft
(B10.BR, H-2k) that was rejected at day 12
posttransplantation (Fig. 1b), thereby confirming that the
tolerant state was donor strain specific.
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Histologic assessment of the allografts revealed a dense
mononuclear leukocytic infiltrate in untreated recipients (Fig. 2a) and less intense
infiltration was present in CTLA/Fc- or IL-15 mutant/Fc-treated
recipients (Fig. 2, b and c) at day 8
posttransplantation. This day was chosen for study because it is the
time at which graft infiltration by host mononuclear leukocytes peaks
in untreated control recipients. In the combined treatment group,
cellular infiltration was dramatically reduced and islet architecture
was well preserved (Fig. 2d). In untreated recipients, dense
graft infiltration by mononuclear leukocytes, composed predominantly of
CD4+ and CD8+ T cells, was
noted (Fig. 3, a and
b). By comparison (and as anticipated), a decrease in graft
infiltration by CD4+ T cells was noted in
recipients treated with CTLA4/Fc (Fig. 3c) while
infiltration by CD8+ T cells was similar to that
detected in untreated mice (Fig. 3d). This result is in
keeping with the hypothesis that costimulation-resistant rejection is a
CD8+ T cell-dependent process. In contrast, in
recipients treated with IL-15 mutant/Fc2a (Fig. 3, e and
f), a decrease in CD8+ T cell
infiltration without a major change in CD4+ T
cell infiltration was evident. Note that recipient mice treated with
both CTLA4/Fc and IL-15 mutant/Fc2a (Fig. 3, g and
h) show a near absence of CD4+ and
CD8+ graft-infiltrating T cells.
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gene
expression was analyzed by RT-PCR at day 8 posttransplantation. As the
constant chain of TCR C is constitutively expressed by T cells, a
substantial decrease in the frequency of graft-infiltrating T cells in
the allograft should be accompanied by a decrease in the intragraft TCR
C gene expression. In recipients treated with IL-15 mutant/Fc2a
or the combination of IL-15 mutant/Fc2a protein and CTLA4/Fc, a
marked decrease in expression of the IL-2 gene and TCR C gene was
observed (data not shown).
To further test the hypothesis that CD8+ T cells
play a major role in costimulation blockade-resistant rejection in this
model, we assessed CTL-selective gene expression events in islet
allografts isolated 8 days posttransplantation (Fig. 4). The granzyme B, perforin, and FasL
genes are uniformly expressed in tissue samples obtained from untreated
recipients or from recipients treated with CTLA4/Fc (Fig. 4). In
contrast, reduced expression of the CTL-selective genes was observed in
recipients treated with IL-15 mutant/Fc2a or in recipients treated
with IL-15 mutant/Fc2a plus CTLA4/Fc (Fig. 4).
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To determine whether treatment with the IL-15 mutant/Fc2a
protein blunts the proliferation of alloreactive
CD8+ T cells in vivo, we labeled splenic
lymphocytes from C57BL/6 CD4-/- mice with CFSE.
The dye-labeled C57BL/6 CD4-/- lymphocytes were
injected i.v. into irradiated untreated or treated (for 3 days) BALB/c
mice. On the third day following adoptive cell transfer, CFSE-stained
leukocytes from hosts were recovered and stained with a biotinylated
anti-CD8 mAb. Since CFSE partitions equally between daughter cells
following cell division, the pattern and frequency of proliferation of
alloreactive CD8+ T cells in vivo can be analyzed
via the CFSE staining pattern (31). In untreated hosts,
23% of CFSE-labeled allogeneic CD8+ T cells
proliferated in the host spleen (Fig. 5b). In CTLA4/Fc-treated
hosts, CD8+ T cells (21%) divide for multiple
generations (Fig. 5b). In contrast, treatment with IL-15
mutant/Fc alone or in combination with CTLA4/Fc markedly inhibited
proliferation of alloreactive CD8+ T cells with
only 15 and 14%, respectively, of CFSE-labeled
CD8+ T cells proliferating in vivo (Fig. 5b). Using syngeneic controls (CFSE-labeled C57BL/6
CD4-/- lymphocytes injected into C57BL/6 mice)
proliferation of CD8+ T cells was not detected
(data not shown). By contrast, when similar experiments were performed
with splenic lymphocytes from C57BL/6
CD8-/-mice to analyze the fate of
CD4+ T cells, we observed that CTLA4/Fc treatment
markedly decreased the frequency of proliferating CFSE-labeled
alloreactive CD4+ T cells in vivo (Fig. 5a). Treatment with IL-15 mutant/Fc2a reduces the
frequency of proliferating CD4+ and
CD8+ T cells, although a lesser effect on
CD4+ T cells as compared with
CD8+ T cells was noted. Similar results were
observed when wild-type C57BL/6 mice were used as donor (data
not shown). IL-15 mutant/Fc2a may represent an important therapeutic
agent capable of controlling the proliferation of alloreactive
CD8+ (costimulation-resistant) T cells.
Proliferation of CD4+, but not
CD8+, T cells is controlled by B7 blockade
agents.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since the molecular targets of costimulation blockade are primarily
expressed upon CD4+, and not upon
CD8+ T cells, the reason that
CD8+ T cells play a prominent role in
costimulation blockade escape rejection process is enigmatic. Studies
using IL-15-/- mice and
IL-15R-/- mice (19, 20) have
emphasized the role of IL-15 in support of the activation and
maintenance of CD8+ T cells (22).
Because costimulation blockade results in diminished expression of IL-2
by CD4+ T cells (7, 9), we
hypothesized that escape from costimulation blockade might represent an
IL-15/IL-15R-dependent process. Moreover, costimulation blockade does
not target epithelial cells, the principle source of IL-15
(23). Thus, we wanted to study the effect of IL-15
mutant/Fc2a on an islet allograft model and its potential to enhance
the effectiveness of costimulatory blockade.
For our experiments, we first chose a partial MHC-mismatch combination
(H-2d–H-2b/d.k) to examine
the effect of the IL-15 mutant/Fc2a. In this islet allograft model,
the administration of IL-15 mutant/Fc2a, a protein that targets the
IL-15R chain expressed upon activated T cells, prevents allograft
rejection in 50% of the recipient mice (Table I
). Identical results
were obtained with CTLA4/Fc administration. It is notable that combined
treatment with CTLA4/Fc and IL-15 mutant/Fc2a produced permanent
engraftment and tolerance in all recipients. Next, we chose to study a
fully MHC-mismatched strain combination and used a recipient strain
(C57BL/6) that is refractory to costimulatory blockade therapy
(15). A modest beneficial effect of CTLA4/Fc treatment was
observed (MST, 30 days vs 13 days in the controls; Fig. 1a)
and prolonged graft survival was evident in IL-15
mutant/Fc2a-treated recipients (Fig. 1a). Treatment with
a control IgG2a protein did not prolong engraftment (MST, 14 days).
Furthermore, combined treatment with CTLA4/Fc and IgG2a does yield an
improvement upon the effect of CTLA4/Fc monotherapy (Fig. 1a). Moreover, the bioavailability of CTLA4/Fc or IL-15
mutant/Fc2a is not altered when the fusion proteins are administered
as combined treatment (CTLA4/Fc half-life = 96 h and IL-15
mutant/Fc2a half-life = 6–8 h). Thus, the beneficial effects
observed with CTLA4/Fc and/or IL-15 mutant/Fc2a are not dependent on
solely the 2a Fc peptide sequence present in the fusion proteins. We
have previously demonstrated that the IL-15 mutant/Fc2a fusion
protein specifically binds to IL-15R but not the common -chain
(28). Binding of the IL-15 mutant/Fc2a was inhibited by
IL-15 but not inhibited by IL-2 or anti-common -chain Abs
(28).
To begin to test our primary hypothesis concerning the role of
IL-15/IL-15R-dependent CD8+ processes in
rejections occurring despite use of CTLA4/Fc, we treated a group of
recipient mice with combined CTLA4/Fc and IL-15 mutant/Fc2a therapy.
Permanent engraftment was obtained in most treated recipients (Fig. 1a). Further analysis demonstrated that these permanently
engrafted mice were rendered specifically tolerant to donor strain
grafts (Fig. 1b). CTLA4/Fc treatment had a far more dramatic
effect upon CD4+ T cell infiltration into the
graft than upon CD8+ T cell infiltration. While
the principal effect of IL-15 mutant/Fc2a treatment in the combined
CTLA4/Fc plus IL-15 mutant treatment group was exacted upon
infiltration of CD8+ T cells into the graft (Fig. 3). The prolongation of islet allograft survival in recipient
mice receiving both CTLA4/Fc and IL-15 mutant/Fc2a was accompanied
by a decrease in T cell infiltration in graft tissues (Fig. 2).
Immunohistologic analysis suggests that the potential benefit of a
combined treatment is related to the drastic decrease of tissue
infiltration by both alloreactive CD4+ and
CD8+ T cells (Fig. 3). Molecular markers for
activated CD8+ T cells, such as CTL genes, have
been identified within the infiltrate of rejecting allografts
(28), and expression of these CTL genes (granzyme B, FasL,
perforin) has been associated with acute renal allograft rejection
(18). It is thus interesting to observe that the
expression of CTL genes are markedly decreased in IL-15
mutant/Fc2a-treated mice (Fig. 4), further attesting to the effect
of IL-15 mutant/Fc2a treatment upon alloactivated
CD8+/CTLs.
To further analyze the effect of costimulation blockade and IL-15
mutant/Fc2a, we studied alloantigen-driven proliferative responses
in vivo using the CFSE dye system. CTLA4/Fc treatment has no effect on
the CD8+ T cell proliferative response to
alloantigen, whereas IL-15 mutant/Fc2a has a potent inhibitory
effect on the proliferation of alloreactive CD8+
T cells (Fig. 5). Because IL-15 is of essential importance for the
homeostasis and proliferation of CD8+ T cells
(22, 23), we cannot exclude the possibility that IL-15
mutant/Fc2a treatment decreases the frequency of proliferating
CD8+ T cell by promoting apoptosis of these
responder cells. In contrast, the alloantigen-driven response of
CD4+ T cells was decreased by either CTLA4/Fc or
IL-15 mutant/Fc2a monotherapy (Fig. 5b). The effect of
CTLA4/Fc upon CD4+ T cells is particularly potent
(Fig. 5a). The IL-15 mutant/Fc2a, but not CTLA4/Fc,
treatment targets CD8+ T cells (Fig. 5).
Consequently combined IL-15 mutant/Fc2a plus CTLA4/Fc treatment
exert additive immunosuppressive effects in controlling allograft
rejection (Fig. 5 and Table I).
We now confirm and extend previous observations that costimulation
blockade treatment reduces the frequency of proliferating alloreactive
CD4+ T cells in an allograft model
(34), but is unable to inhibit the proliferation of
alloreactive CD8+ T cells in vivo
(13). The resistance of CD8+ T cells
to costimulation blockade is related to the inability of CTLA4/Fc
(35, 36) and anti-CD154 mAb (36)
treatments to control alloactivated CD8+ T cells
and is probably related to the restricted expression of CD28 and CD154
to CD4+ T cells (37). To obtain
prolonged engraftment of heart allografts, treatment with a
nondepleting anti-CD4 mAb alone is not sufficient. The successful
use of soluble IL-15 receptor -chain proteins as an adjunct to
anti-CD4 treatment (19) is consistent with our
hypothesis that blocking both
CD4+- and
IL-15/IL-15R-dependent CD8+ T cell activation is
required to gain long-term graft acceptance.
IL-15 mutant/Fc2a treatment mediates important effects on allograft
survival that are exerted, at least in part, through the inhibition of
the activation and proliferation of alloreactive
CD8+ T cells (
Figs. 3–5 and Table I). This
finding emphasizes the role of the IL-15/IL-15R pathway as an important
element in the rejection process. Moreover, we have recently noted that
the initial five to six waves of the T cell proliferation to
alloantigens in vivo are IL-15, not IL-2, dependent (38).
The induction of permanent tolerance is still an elusive goal in
clinical organ transplantation. By targeting
IL-15/IL-15R+ cells to prevent costimulation
blockade-resistant rejection, IL-15 mutant/Fc2a appears to provide a
promising new agent capable of interrupting allograft rejection
mediated by CD8+ T cells. The combined effects of
costimulation blockade plus targeting the IL-15/IL-15R pathways to
target both CD4+ and CD8+ T
cells may represent an effective approach to achieve permanent
tolerance.
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Acknowledgments |
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Footnotes |
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2 Current address: Seoul National University, Boraman Hospital, 195 Shindaebang-dong, Tongjak-ju, Seoul, 156-012, Korea.
3 Current address: Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, Warsaw, Poland.
4 Address correspondence and reprint requests to Dr. Terry B. Strom, Division of Immunology, Beth Israel Deaconess Medical Center, Research North, Room 380, P.O. Box 15707, Boston, MA 02215. E-mail address: tstrom{at}caregroup.harvard.edu
5 Abbreviations used in this paper: KO, knockout; FasL, Fas ligand; MST, mean survival time.
Received for publication September 12, 2000. Accepted for publication July 10, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain enhances cardiac allograft survival. J. Immunol. 165:3444. chain of the IL-2 receptor. EMBO J. 14:3654.[Medline]
and chains of the IL-2 receptor by a novel cytokine IL-15. EMBO J. 13:2822.[Medline]
-chains on human peripheral blood mononuclear cells and effect of immunosuppressive drugs on receptor expression. J. Immunol. 157:2813.[Abstract]
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P. H. Horne, M. A. Koester, K. Jayashankar, K. E. Lunsford, H. L. Dziema, and G. L. Bumgardner Disparate Primary and Secondary Allospecific CD8+ T Cell Cytolytic Effector Function in the Presence or Absence of Host CD4+ T Cells J. Immunol., July 1, 2007; 179(1): 80 - 88. [Abstract] [Full Text] [PDF]
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J. Zhang-Hoover, P. Finn, and J. Stein-Streilein Modulation of Ovalbumin-Induced Airway Inflammation and Hyperreactivity by Tolerogenic APC J. Immunol., December 1, 2005; 175(11): 7117 - 7124. [Abstract] [Full Text] [PDF]
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C. Domenig, A. Sanchez-Fueyo, J. Kurtz, S. P. Alexopoulos, C. Mariat, M. Sykes, T. B. Strom, and X. X. Zheng Roles of Deletion and Regulation in Creating Mixed Chimerism and Allograft Tolerance Using a Nonlymphoablative Irradiation-Free Protocol J. Immunol., July 1, 2005; 175(1): 51 - 60. [Abstract] [Full Text] [PDF]
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S. Ferrari-Lacraz, E. Zanelli, M. Neuberg, E. Donskoy, Y. S. Kim, X. X. Zheng, W. W. Hancock, W. Maslinski, X. C. Li, T. B. Strom, et al. Targeting IL-15 Receptor-Bearing Cells with an Antagonist Mutant IL-15/Fc Protein Prevents Disease Development and Progression in Murine Collagen-Induced Arthritis J. Immunol., November 1, 2004; 173(9): 5818 - 5826. [Abstract] [Full Text] [PDF]
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M. D. Vu, F. Amanullah, Y. Li, G. Demirci, M. H. Sayegh, and X. C. Li Different Costimulatory and Growth Factor Requirements for CD4+ and CD8+ T Cell-Mediated Rejection J. Immunol., July 1, 2004; 173(1): 214 - 221. [Abstract] [Full Text] [PDF]
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G. Demirci, F. Amanullah, R. Kewalaramani, H. Yagita, T. B. Strom, M. H. Sayegh, and X. C. Li Critical Role of OX40 in CD28 and CD154-Independent Rejection J. Immunol., February 1, 2004; 172(3): 1691 - 1698. [Abstract] [Full Text] [PDF]
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G. Demirci, W. Gao, X. X. Zheng, T. R. Malek, T. B. Strom, and X. C. Li On CD28/CD40 Ligand Costimulation, Common {gamma}-Chain Signals, and the Alloimmune Response J. Immunol., May 1, 2002; 168(9): 4382 - 4390. [Abstract] [Full Text] [PDF]
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T. R. Jones, J. Ha, M. A. Williams, A. B. Adams, M. M. Durham, P. A. Rees, S. R. Cowan, T. C. Pearson, and C. P. Larsen The Role of the IL-2 Pathway in Costimulation Blockade-Resistant Rejection of Allografts J. Immunol., February 1, 2002; 168(3): 1123 - 1130. [Abstract] [Full Text] [PDF]
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) or recipients treated with a control IgG2a
protein (1.5 µg) rejected allografts with a MST of 15 days and 14
days, respectively (
). Graft rejection was delayed by 30 days in
recipients treated with CTLA4/Fc (
), CTLA4/Fc plus IgG2a (
), or
IL-15 m/Fc
), whereas in recipients receiving the combined
treatment, the MST was of 120 days (•). b, Second
islet transplantation. Nephrectomy of the left kidney was performed on
five recipients 150 days after transplantation. Then four
nephrectomized mice received a second BALB/c islet allograft under the
right renal capsule (following the same procedure) without further
immunosuppression. All of them accepted the second transplant for more
than 50 days, demonstrating a state of tolerance (•). One mouse
received a third part islet allograft (B10. BR, H-2k; 
