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2a Protein Blocks Delayed-Type Hypersensitivity1
*
Department of Medicine, Harvard Medical School, Divisions of
Immunology and
Gastroenterology, Beth Israel Deaconess Medical Center and
§
Renal Division, Brigham and Women’s Hospital, Boston, MA 02215
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-15/IL-15R differ. The development
of agents targeting the receptor and signaling elements of IL-15 may
provide a new perspective for treatment of diseases associated with
expression of IL-15/IL-15R. We designed, genetically constructed, and
expressed a receptor site-specific IL-15 antagonist by mutating
glutamine residues within the C terminus of IL-15 to aspartic acid and
genetically linked this mutant IL-15 to murine Fc
2a. These
mutant IL-15 proteins specifically bind to the IL-15R, competitively
inhibit IL-15-triggered cell proliferation, and do not activate the
STAT-signaling pathway. Because the receptor site-specific antagonist
IL-15 mutant/Fc2a fusion proteins had a prolonged
t1/2 in vivo and the potential for
destruction of IL-15R+ leukocytes, we examined the
immunosuppressive activity of this agent. An IL-15 mutant/Fc2a
fusion protein markedly attenuated Ag-specific delayed-type
hypersensitivity responses and decreased leukocyte infiltration within
the delayed-type hypersensitivity sites. These findings suggest that 1)
IL-15/IL-15R+ cells are crucial to these T cell-dependent
immune responses, and 2) treatment with IL-15 mutant/Fc2a protein
may ameliorate T cell-dependent immune/inflammatory diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF- synthesis (2, 3, 4, 5, 6, 7). The production of
IL-15 is regulated at transcriptional and posttranscriptional levels
(4). Unlike IL-2, IL-15 transcripts are not detected in resting or
activated T cells (4, 5). Nonetheless, IL-15 expression is associated
with exacerbations of autoimmune diseases such as rheumatoid arthritis
(8, 9, 10), inflammatory bowel disease (11), and allograft rejection
(12, 13).
The IL-15R comprises three elements; IL-15R, IL-2Rß, and IL-2R
(also called common or c) (14, 15, 16). Thus, the
IL-2R and IL-15R share two components. IL-15R mRNA is expressed in a
wide range of cell types, including activated T cells, activated NK
cells, activated B cells, activated macrophages, activated vascular
endothelial cells, as well as thymic and bone marrow stromal cells.
IL-15R mRNA also has wide range of tissue expression such as liver,
heart, spleen, lung, and skeletal muscle (4, 16). The development of
agents targeting IL-15R may provide a new perspective for the treatment
of immune and inflammatory diseases.
Cytokines possess a very high affinity for their receptor, but their short lives and agonist activity, triggering activation of receptor-bearing target cells, limit or preclude their utility as receptor site antagonists or a vehicle for targeting cytocidal agents to cytokine receptor-bearing cells without transiently stimulating the target cells. The relatively short in vivo t1/2 of many cytokines hamper their therapeutic efficacy and require frequent injection or constant administration (1). To overcome the problem associated with the short t1/2, we and others have generated long-lived cytokine/IgG-related fusion proteins (17, 18, 19). Depending upon the desired application, the Fc region can be chosen to express or preclude cytocidal activity against the target cells (17, 19).
Petitt et al. (20) demonstrated that mutation of the glutamine at
residue 108 in human IL-15 to serine creates an IL-15R site-specific
antagonist. In our laboratory, an IL-15 antagonist was also constructed
by replacing the codons for the C-terminal glutamine amino acid
residues with codons for aspartic acid (i.e., Q101 and Q108) and we
developed a strategy for selective targeting high affinity
IL-15R-bearing cells by use of IL-15 mutant/Fc2a fusion proteins.
IL-15 mutant/Fc2a proteins have a high affinity, receptor
site-specific IL-15R binding, and antagonist properties; fail to
activate the STAT system; and possess a prolonged
t1/2 in vivo. Treatment with the IL-15
mutant/Fc2a fusion protein markedly attenuates Ag-specific
DTH4 responses and cellular
infiltration within the DTH sites. These findings suggest that IL-15
and/or IL-15R+ cells are crucial for, at least, some
Ag-specific T cell-mediated immune response in vivo. Hence, IL-15
mutant/Fc2a proteins may provide therapeutic benefit for certain T
cell-dependent immune diseases and other IL-15-rich inflammatory
states.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2a
Human IL-15 and murine Fc2a cDNAs were generated from
mRNA extracted from PHA-stimulated human PBMCs and IgG2a-secreting
hybridoma (American Type Cell Culture (ATCC) HB129, Rockville, MD),
respectively, using reverse-transcriptase MMLV-RT (Life Technologies,
Gaithersburg, MD) and synthetic oligo(dT) oligonucleotides (Life
Technologies). The IL-15 mutant cDNAs were designed to target select
glutamine codons of human IL-15 (Q101 and Q108) for mutation to
aspartic acid sequences by PCR-assisted site-directed mutagenesis. For
the construction of mutant plasmids, a 322-bp cDNA fragment encoding
mature human IL-15 with relevant mutations at positions 101 and 108 was
amplified by PCR utilizing synthetic sense:
5'-GAAGCTTGAACTGGGTGAATGTAATAAGT-3' (HindIII site
plus bases) and antisense oligonucleotides corresponding to the
C-terminal fragment of human IL-15, followed by a BamHI site
(the mutated codons are underlined and in bold):
5'-TCTGGGATCCGAAGTGTTGATGAACATGTCGACAATATGTACAAAACTGTCCAAAAAT-3'.
Synthetic oligonucleotides used for the amplification of the Fc2a
domain cDNA change the first codon of the hinge region from Glu to Asp
to create a unique BamHI site spanning in the first codon of
the hinge and introduce a unique XbaI site 3' to the
termination codon. Ligation of cytokine and Fc2a components in the
correct translational reading frame yields a 1059-bp-long open frame
encoding a single 353-amino-acid polypeptide. The mature secreted
homodimeric IL-15 mutant/Fc2a is predicted to have a m.w. of 80 kDa,
exclusive of glycosylation. Proper genetic construction of IL-15
mutant/Fc2a was confirmed by DNA sequence analysis following cloning
of the fusion genes into the eukaryotic expression plasmid pSecTag
(Invitrogen, San Diego, CA). This plasmid carries a CMV promoter, IgG
leader sequence, and a gene for selection against Zeocin
(Invitrogen).
Expression and purification of IL-15 mutant/Fc2a
Plasmids carrying fusion genes were transfected into NS.1 cells
(ATCC) by electroporation (1.5 kV/3 µF/0.4 cm/PBS) and selected in
serum-free Ultraculture media (BioWhittaker, Walkersville, MD)
containing 100 µg/ml Zeocin. After subcloning, high producing clones
were selected by screening supernatants for IgG2a by ELISA. IL-15
mutant/Fc2a fusion proteins were purified from culture supernatant
by protein A-Sepharose affinity chromatography (Pharmacia, Piscataway,
NJ), followed by dialysis against PBS and 0.22-µm filter
sterilization. Purified proteins were stored at -20°C until use. The
size and specificity of purified IL-15 mutant/Fc2a were confirmed by
SDS-PAGE under reducing (+DTT) and nonreducing (-DTT) conditions,
followed by Western blot analysis using polyclonal anti-human IL-15
(PeproTech, Rocky Hill, NJ) and anti-murine IgG2a Abs (PharMingen,
San Diego, CA).
Determination of IL-15 mutant/Fc2a circulating
t1/2
The serum concentration of IL-15 mutant/Fc2a was
determined at various time points following a single bolus i.v.
injection of the fusion protein that was administered to 8- to
10-wk-old BALB/c mice (The Jackson Laboratory). Serial 100-µl blood
samples were obtained by retroorbital bleeding at intervals of 0.1, 6,
24, 48, 72, and 96 h after administration. Measurements of IL-15
mutant/Fc2a were made by ELISA using rabbit anti-human IL-15 Ab
as the capture Ab and horseradish peroxidase-conjugated anti-mouse
IgG2a mAb as the detection Ab (PharMingen). This assured that the ELISA
was specific for the IL-15 mutant/Fc2a protein, and not IL-15 or
mouse IgG2a.
Proliferation assays
IL-3-dependent BAF-BO3 cells expressing IL-2Rß chains were
washed twice to remove the growth factor and starved for 6 h in
RPMI 1640 medium supplemented with 1% FCS, penicillin, and
streptomycin. Cells were then plated (2 x 104
cells/well) and cultured for 48 h at 37°C with medium alone or
medium supplemented with IL-3-rich supernatants from WEHI cells,
rhIL-2, or rhIL-15 in an atmosphere containing 5% CO2.
Following this incubation, cells were pulsed for 6 h with 1 µCi
[3H]TdR and harvested onto Whatman 934-AH glass
microfiber filters using a PHD cell harvester (Cambridge Technology,
Cambridge, MA). Cell-associated [3H]TdR was measured
using a Beckman LS 2800 scintillation counter (Beckman, Fullerton, CA).
To probe for receptor site-specific antagonist activity, growth factors
(IL-3-rich media, rhIL-2, or rhIL-15) were added simultaneously with
the indicated concentrations of IL-15 mutant/Fc2a proteins. BAF-BO3
cells were then harvested, and cell-associated radioactivity was
measured by scintillation counting, described as above. BAF-BO3 cells
cultured with IL-15 mutant/Fc2a proteins for 3 days were stained
with trypan blue to determine cell viability. No evidence of cell
toxicity was observed in IL-15 mutant/Fc2a-treated cells in
comparison with controls.
Immunoblotting for STAT proteins
IL-3-dependent BAF-BO3 cells expressing IL-2Rß chains were
washed twice to remove the growth factor and starved for 6 h in
RPMI 1640 medium supplemented with 1% FCS, penicillin, and
streptomycin. Cells were washed again, resuspended in RPMI 1640
(107 cells/ml), and stimulated with medium alone, or
medium supplemented with either 50 U/ml of rhIL-2, 10 ng of rhIL-15, or
IL-15 mutant/Fc2a proteins. Following interaction with these
proteins for 2 min at 37°C, the cells were washed with ice-cold PBS
and lysed for 15 min on ice in 50 µl of buffer containing NaCl (150
mM), Nonidet P-40 (1%), Tris-HCl, pH 7.5 (25 mM),
Na3VO4 (1 mM), PMSF (1 µM), leupeptin (10
µg/ml), and aprotinin (10 µg/ml). Cellular debris was removed by
centrifugation, and proteins present in the supernatants of these cell
lysates were separated on 7.5% SDS-PAGE under reducing conditions and
transferred onto polyvinylidene difluoride membranes. The membranes
were blocked for 1 h at room temperature in buffer containing
Tris-HCl (20 mM, pH 7.5), NaCl (150 mM), Tween-20 (0.1%), and BSA
(3%), and then incubated for 1 h with phosphospecific STAT3 Ab
(New England Biolabs, Beverly, MA). The membrane was washed and
developed using the SuperSignal Western blotting kit (Pierce, Rockford,
IL), according to the manufacturer’s protocol. For subsequent
staining, the membranes were incubated at 50°C for 20 min in
stripping buffer containing 100 mM 2-ME, 2% SDS, and 62.5 mM Tris-HCl,
pH 6.7, followed by a 1-h incubation in blocking buffer, and
immunodetection was performed using anti-STAT3 Ab (Santa Cruz
Biotechnology, Santa Cruz, CA). For the detection of phosphorylation of
STAT5 proteins, cell lysates from BAF-BO3 cells expressing IL-2Rß,
stimulated described as above, were separated on SDS-PAGE. After
transfer onto polyvinylidene difluoride membranes, immunodetection of
phosphorylated proteins was performed using horseradish
peroxidase-conjugated anti-phosphotyrosine Ab (Zymed, South San
Francisco, CA). This membrane was blotted again using anti-STAT5 Ab
(Transduction Laboratories, Lexington, KY) after stripping described as
above.
Cell staining for flow cytometry
BAF-BO3 cells expressing IL-2Rß (106
cells/tube) were washed twice with ice-cold PBS/0.02% sodium azide.
After blocking with control mouse IgG (PharMingen), cells were
incubated in medium alone (control) or medium containing IL-15
mutant/Fc on ice for 30 min, washed with PBS, and incubated for 30 min
with FITC-conjugated goat anti-mouse Fc Ab (Pierce). To define the
receptor-ligand specificity, molar excess rhIL-2, rhIL-15, or rat
anti-mouse IL-2R Ab (4G3/3E12; kind gift of Dr. Thomas R. Malek,
Miami, FL) was added to medium containing IL-15 mutant/Fc2a. Cells
were stained with FITC-conjugated goat anti-mouse Fc Ab. Cell
staining was analyzed using FACScan (Becton Dickinson, Mountain View,
CA) and CellQuest software.
DTH responses
BALB/c (The Jackson Laboratory) mice were sensitized to
methylated BSA (MBSA; Sigma, St. Louis, MO) by an intradermal injection
of 50 µl of 5 mg/ml MBSA in CFA (Sigma) at two sites on the abdomen.
Eight days after immunization, the mice were rechallenged by injection
of 20 µl of 5 mg/ml MBSA into one rear footpad, while the other rear
footpad received a comparable volume of PBS. Measurements of footpad
swelling were taken at 24, 48, and 72 h after challenge by use of
two different micrometers (Mitutoyo, Tokyo, Japan, and LS Starrett,
Athol, MA). The magnitude of the DTH responses was determined as the
differences in footpad thickness between the Ag- and PBS-injected
footpads. DTH responses were measured in a blinded fashion, in which
measurements were obtained by an individual who did not know the
treatment protocol for each subject. Treatment protocols were: 1) i.p.
injection of 1.5 µg of either IL-15 mutant/Fc2a or mouse
IgG2a daily, starting 30 min before the rechallenge with MBSA and
continued for 3 days with or without concomitant cyclosporine (CsA;
Novartis, Hanover, NJ) (2 mg/kg of CsA i.p. as loading dose, and 0.5
mg/kg on the following 2 days), or 2) i.p. injection of 1.5 µg of
either IL-15 mutant/Fc2a or mouse IgG2a daily, starting 30 min
before the initial challenge of MBSA and continued daily for 8 days.
For histologic examination of the DTH reaction sites in the first
treatment protocol, soft tissue samples from the foot were collected at
12 and 24 h after the second MBSA challenge.
Immunohistochemistry
Cryostat tissue sections (6 µm) were placed on slides coated with poly(L-lysine) (Sigma) and fixed for 10 min in 95% ethanol at 4°C. Endogenous peroxidase activity, present in tissue sections, was blocked by treatment with 0.6% H2O2 and 0.2% NaN3 for 10 min. Tissue sections were then incubated for 15 min each in avidin solution, followed by biotin solution (Vector Laboratories, Burlingame, CA) to block endogenous biotin. Nonspecific IgG binding was prevented by pretreatment of tissue sections for 30 min with 10% normal rabbit serum and 10% BSA. Tissue sections were then incubated overnight with rat anti-mouse primary Ab (10 µg/ml) in 1% BSA at 4°C. Bound primary Ab was then labeled with rabbit anti-rat IgG conjugated with biotin for 1 h at room temperature. The sections were then incubated with avidin-biotin-horseradish peroxidase complex (Vectastain ABC reagent; Vector Laboratories) for 1 h at room temperature. Diaminobenzidine substrate solution (Vector Laboratories) was then added to tissue sections, resulting in a brown color at sites of immunoenzymatic labeled Ag. Tissue sections were then counterstained with hematoxylin to detect cell nuclei. Positively stained cells were counted in 10 randomly selected fields (each 100 µm2). The following primary Abs were used for immunostaining: rat anti-mouse CD4 IgG2a clone RM4-5 (PharMingen) and rat anti-mouse F4/80 IgG2b (prepared from hybridoma supernatant, ATCC HB198). The negative isotype control Abs used were rat IgG2a clone R35-95 and rat IgG2b clone R35-38 (PharMingen). The secondary Ab used for all immunostaining was rabbit anti-rat IgG conjugated with biotin (Vector Laboratories).
Statistics
Student’s t test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2a fusion proteins
In previous studies, we demonstrated that FLAG-HMK-IL-15
specifically binds to IL-15R expressed on PHA-activated PBMCs (21) and
T84 colonic cryptlike intestinal epithelial carcinoma cells (22).
Mutations targeting glutamine residues localized in the C-terminal
-helix of human IL-15 do not destroy the ability of these
FLAG-HMK-IL-15 mutant proteins to bind to IL-15R (manuscript
submitted5). In keeping
with the observations of Pettit et al. (20), an IL-15-related glutamine
to aspartic acid mutant, i.e., FLAG-HMK-IL-15 Q101D,Q108D proteins,
specifically and competitively block IL-15-triggered cell proliferation
(data not shown). This FLAG-HMK-IL-15 Q101D,Q108D mutant protein is an
antagonist for rhIL-15-triggered proliferation. As the FLAG epitope is
immunogenic, and the t1/2 of unmodified cytokine
is short (1), these features limit therapeutic application. Thus, we
developed an IL-15 mutant/Fc2a fusion protein to provide a receptor
site-specific antagonist with a prolonged circulating
t1/2 and cytocidal potential. To confirm the
molecular size and the cytokine/isotype specificity, the
affinity-purified fusion protein was characterized by Western blot
analysis following 12% SDS-PAGE. As shown in Figure 1
, the IL-15 mutant/Fc2a fusion
proteins migrated under reducing (+DTT) conditions as a single species
at a molecular size of 46 kDa (Fig. 1, lanes 1 and
3). Under nonreducing (-DTT) conditions, each IL-15
mutant/Fc2a fusion protein runs as a single species at a molecular
size of 95 kDa (Fig. 1, lanes 2 and 4),
which indicates that the IL-15 mutant/Fc2a fusion protein is
expressed as a homodimer. Moreover, the IL-15 mutant/Fc2a fusion
protein is immunoreactive with both anti-human IL-15 Ab (Fig. 1, lanes 1 and 2) and anti-mouse IgG2a Ab
(Fig. 1, lanes 3 and 4), confirming the
cytokine and isotype specificity of the IL-15 moiety and Fc2a
domain, respectively.
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2a fusion
protein binds to IL-15R expressed upon IL-2Rß+ BAF-BO3
cells (Fig. 2A). The
specificity of the IL-15 mutant/Fc2a binding for IL-15 binding sites
was established through a study in which the binding of the IL-15
mutant/Fc2a to target cells was blocked by provision of a molar
excess of rhIL-15 (Fig. 2B) and not inhibited by a
molar excess of rhIL-2 (Fig. 2C) or 4G3/3E12 rat
anti-mouse IL-2R Ab (Fig. 2D).
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2a fusion proteins fail to support cell
proliferation and to trigger tyrosine phosphorylation of STAT3 and
STAT5 proteins
The impact of mutation of the C-terminal glutamine residues and
linking of the mutant IL-15 to the Fc domain on the biologic activity
of IL-15 was probed. The IL-15 mutant/Fc2a fusion protein fails
to support the proliferation of IL-15-sensitive IL-2Rß+
BAF-BO3 cells. Furthermore, simultaneous addition of the mutant IL-15
protein blocks rhIL-15-driven cell proliferation in dose-dependent
manner (Fig. 3), while rhIL-2- or
IL-3-rich medium-dependent cell proliferation is not inhibited by the
addition of IL-15 mutant/Fc2a, even in excess amount of fusion
proteins (Fig. 3).
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2a fusion protein to trigger tyrosine
phosphorylation of STAT3 and STAT5 proteins in IL-2Rß+
BAF-BO3 cells. Unlike rhIL-15, IL-15 proteins bearing the Q101D and
Q108D mutations fail to stimulate tyrosine phosphorylation of STAT3 and
STAT5 (Fig. 4), thereby linking the
failure of tyrosyl phosphorylation of STAT3 and STAT5 proteins with the
failure to trigger target cell proliferation.
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2a fusion protein studied
in vivo: circulating t1/2
We determined the circulating t1/2 of the
IL-15 mutant/Fc2a fusion protein using a unique dual-probe ELISA
that detects the IL-15 mutant/Fc2a fusion protein, but not IL-15 nor
mouse IgG2a. The circulating t1/2 of the IL-15
mutant/Fc2a fusion protein was 6 h (Fig. 5). Thus, the t1/2
of the IL-15 mutant/Fc2a fusion protein is prolonged in comparison
with the t1/2 of unmodified IL-15, which is 2 to
3 min (20).
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2a fusion proteins block DTH in normal mice
To determine whether IL-15 mutant/Fc2a treatment blocks T
cell-dependent in vivo responses to an Ag, DTH responses were
evaluated. After the initial immunization with MBSA, mice were treated
with either the IL-15 mutant/Fc2a fusion protein or mouse IgG in
control group starting just before rechallenge of MBSA with or without
concomitant CsA. As shown in Table I,
control mouse IgG-treated mice mounted a brisk DTH response to a
rechallenge of MBSA. Treatment with CsA and control IgG did not
markedly attenuate the DTH response, while treatment with IL-15
mutant/Fc2a protein blocked the DTH response. This reduction in DTH
was reflected by a decreased influx of macrophages (Fig. 6) and CD4+ T cells (Fig. 7) within the footpad dermis in IL-15
mutant/Fc2a-treated mice vs control mice. Combined treatment with
IL-15 mutant/Fc2a plus CsA reduced the DTH response synergistically
and further suppressed the cellular infiltration (Table I; Figs. 6 and 7).
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2a, we tested the
efficacy of prolonged treatment with IL-15 mutant/Fc2a proteins.
IL-15 mutant/Fc2a was administered just before the initial challenge
of MBSA and continued daily until the day of Ag rechallenge
(Materials and Methods). Control mouse
IgG-treated mice showed a brisk DTH response to rechallenge of MBSA,
while the DTH responses in mice given IL-15 mutant/Fc2a were
markedly attenuated (control mouse IgG-treated vs IL-15
mutant/Fc2a-treated mice: 0.86 ± 0.084 mm vs 0.23 ±
0.143 at 24 h after rechallenge, 0.53 ± 0.174 vs 0.13
± 0.052 at 48 h, and 0.33 ± 0.041 vs 0.09 ± 0.053 at
72 h, respectively; mean ± SD, p < 0.01; 7
mice/each group). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix bundle
family of cytokines that possess T cell growth-factor activity (2, 6).
In contrast to IL-2, a T cell product, IL-15 mRNA is expressed by a
wide variety of cells, including macrophages, B cells, thymic,
activated vascular endothelial cells, and bone marrow stromal cells, as
well as tissues such as liver, heart, spleen, lung, and skeletal muscle
(4, 6). Despite their differing cellular origins, IL-15 and IL-2
exert overlapping activities due to their shared ß- and -chain
receptor components (3, 25). While the expression of IL-2R and
IL-15R upon mononuclear leukocytes is limited to recently activated
cells, the tissue distribution of the unique IL-15R component on
nonimmune cells suggests that IL-15 has activity outside the immune
system, such as anabolic activities on myocytes (26) and increasing
transepithelial resistance on colonic epithelial cells (22).
IL-15 expression is associated with exacerbations of rheumatoid
arthritis (8, 9, 10), sarcoidosis (27), and inflammatory bowel disease
(11), as well as allograft rejection (12, 13). Because the importance
of IL-15/IL-15R+ cells to these immune/inflammatory
disease states is not certain, we sought to target IL-15R+
cells with a very high affinity receptor site-specific antagonist
possessing a prolonged circulating t1/2 and the
potential for cytocidal targeting of IL-15R+ cells. In this
study, we report the design and properties of an IL-15 mutant/Fc2a
(Q101D,Q108D) immunoligand protein (Fig. 1
) that 1) specifically binds
with high affinity to IL-15R (Fig. 2), 2) specifically inhibits
IL-15-stimulated proliferative responses (Fig. 3), 3) fails to activate
STAT-signaling pathway (Fig. 4), and 4) has a prolonged in vivo serum
t1/2 of 6 h (Fig. 5). Importantly, the
potential therapeutic value of the IL-15 mutant/Fc2a is hinted by
the attenuation of T cell-dependent Ag responses (DTH) (Table I; Figs. 6 and 7).
The in vitro binding and proliferative results for IL-15 mutant/Fc2a
parallel those reported for bacterially expressed IL-15 mutant proteins
(manuscript submitted5) (20). The IL-15 mutant/Fc2a
blocked cell proliferation triggered by rhIL-15, but not rhIL-2 (Fig. 3). Even excess amounts of IL-15 mutant/Fc2a fusion protein failed
to inhibit IL-2-driven cell proliferation, while both rhIL-2- and
rhIL-15-dependent IL-2Rß+ BAF-BO3 cell proliferation was
blocked by 4G3/3E12 rat anti-mouse IL-2R (data not shown). In
addition, binding of this mutant protein was not blocked by different
growth factors, even though they share occupation of certain receptor
subunits (Fig. 2). Combining the flow-cytometric analysis with cell
proliferation results, human IL-15 and the IL-15-related mutant protein
bind to mouse IL-15R. Therefore, the IL-15 mutant/Fc2a protein can
be used to distinguish IL-15 from IL-2-mediated responses. Using
IL-15-sensitive cells, we now demonstrate that IL-15 mutant/Fc2a
fails to stimulate phosphorylation of STAT3 and STAT5 proteins that are
critical to IL-15 intracellular signaling (23, 24). Clearly, glutamine
residues localized in the C-terminal -helix of the IL-15 molecule
are crucial for STAT protein activation, which is a critical component
of the intracellular signaling cascade leading to IL-15-mediated
proliferation. Given the similar three-dimensional structures of IL-15
and IL-2 and the fact that a C-terminal glutamine in IL-2 is
responsible for IL-2R chain binding (28), it is reasonable to
speculate that Q101D,Q108D IL-15 mutant/Fc2a proteins cannot
transduce signals through the IL-2R chain.
Genetic linkage of IL-15 to Fc enhanced the
t1/2 of the IL-15 moiety (Fig. 5), as previously
reported for fusion proteins involving IL-2 (18), IL-10 (19), and IL-4
(17). The t1/2 of 6 h for IL-15
mutant/Fc2a is not as long as the 33-h t1/2
for IL-10/Fc2a molecule (19), perhaps due to the larger tissue
distribution of IL-15R than IL-10R. A second advantage of immunoligand
construction is the opportunity to manipulate the Fc backbone to
produce, as previously described, lytic and nonlytic forms of molecules
(17, 19, 29). The known complement fixation and Ab-dependent cell
cytotoxicity binding sites of the Fc moiety can be mutated to generate
immunoligands that are nonlytic (19). In these studies, we used the
native Fc2a backbone to create the IL-15 mutant/Fc2a fusion
protein. This sequence provides longevity (1) and the ability to
activate complement on receptor-bearing leukocytes.
This laboratory has reported previously that in vivo
administration of an IL-2 diphtheria toxin-related fusion protein
blocks DTH (30). As IL-15/IL-15R mRNAs are expressed upon activated
lymphocytes as well as tissues targeted by T cell-mediated immune
reactions, we postulated that IL-15R-targeted treatment, as previously
documented for IL-2R-targeted treatment (30), would also inhibit
Th1-dependent in vivo DTH responses (31, 32, 33).
Commensurate with an attenuation in inflammation (e.g., footpad
swelling) (Table I), IL-15 mutant/Fc2a treatment reduced the
intralesional infiltration of macrophages and CD4+ T cells
(Figs. 6 and 7). Indeed, treatment with the IL-15 mutant/Fc2a proved
more potent than a standard dose of CsA. Combined treatment with CsA
plus IL-15 mutant/Fc2a synergistically inhibited the DTH reaction
(Table I; Figs. 6 and 7). Although the mechanism by which the IL-15
mutant protein blocks T cell-dependent DTH responses was not directly
addressed in this work, we speculate that IL-15R site antagonism and/or
elimination of IL-15R+ cells account for the effectiveness
of IL-15 mutant protein treatment. Since the number of
IL-15R+ (or IL-2R-positive) cells within the inflammatory
lesion is very small, we will determine whether cell lysis is
responsible, at least in part, for diminishing the inflammatory
response by comparing the effects of IL-15 mutant lytic and nonlytic Fc
fusion proteins.
This report characterizes the binding and function of an
antagonist-type IL-15 mutant/Fc2a and demonstrates that targeting of
IL-15R can abrogate an in vivo Th1 response (DTH). Hence, based on the
inhibition of DTH, we suggest that IL-15 mutant/Fc2a protein offers
therapeutic promise as an agent for the treatment of Th1-type
autoimmune diseases, organ transplantation, and other T cell-dependent
disease processes. In short, we have constructed a novel long-lived
IL-15 mutant/Fc2a molecule whose use may aid in determining the
roles of IL-15 and IL-15R+ cells in certain immune and
inflammatory disease states.
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Footnotes |
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2 Current address: Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, Warsaw, Poland.
3 Address correspondence and reprint requests to Dr. Terry B. Strom, Division of Immunology, Beth Israel Deaconess Medical Center, Research North, P.O. Box 15707, Boston, MA 02215. E-mail address:
4 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; CsA, cyclosporine; FLAG, International Biotechnologies-Kodak trade name for marker octapeptide N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C; HMK, heart muscle kinase recognition site; MBSA, methylated bovine serum albumin; rh, recombinant human.
5 Y. S. Kim, D.-W. Chae, Y. Nosaka, A. C. Stevens, T. B. Strom, and W. Maslinski. Certain substitutions at the Glu108 residue of human IL-15 create receptor site specific antagonist proteins. Submitted for publication.
Received for publication November 10, 1997. Accepted for publication February 10, 1998.
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