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Departments of
* Antibody Development,
Preclinical Development,
Preclinical Discovery, and
Protein Development, Human Genome Sciences, Rockville, MD, 20850; and
¶ Division of Rheumatology, Department of Medicine, Los Angeles County and University of Southern California Medical Center and Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to BLyS, the role of APRIL is less clear. Some investigators have shown that APRIL may act as a costimulator for T and B cell activation and as an inducer of tumor cell growth (17), while others have shown it can function as a proapoptotic factor (18). The role of APRIL in autoimmune disease remains to be determined.
TNF ligands have been shown to form trimeric structures composed of three monomers (1). It was initially proposed that ligand homotrimers would cause trimerization of their receptor, but recent studies (19) point to the existence of preassembled receptors. Furthermore, new findings suggest that BLyS may form very large virus-like structures composed of multiple homotrimers (20). This cluster formation is mediated by a "flap" region that is not present in APRIL. How these large oligomers would interact with their receptors has yet to be elucidated. These results are not consistent with two other reports that found that BLyS exists only as a trimer and not as an oligomer (4, 21).
Even though most ligands of the TNF superfamily are known to be
homotrimers, heterotrimeric molecules have been described. LT
and
LT
can form two types of membrane-bound complexes:
LT12 and
LT21, which bind
different receptors (LTR and TNFR1/2, respectively; Refs.
22, 23, 24). Based on the phenotype of LT transgenic and
LT null mice, it appears that LT-induced inflammation is
primarily due to interaction of the LT3
homotrimer with TNFR1, whereas the
LT12 heterotrimeric
complex plays an important role in lymphoid organ development.
While investigating the regulation of APRIL and BLyS expression in
autoimmune diseases, we identified two BLyS-binding mAbs that detected
different levels of BLyS in autoimmune serum samples, but recognized
recombinant human BLyS equally well. This observation suggested that
BLyS might exist in alternative forms in human serum and prompted us to
evaluate whether, like LT and , BLyS might form heterotrimers
with its close homolog APRIL.
In this study, we show that APRIL and BLyS, when coexpressed, can indeed form biologically active heterotrimeric complexes that circulate in the serum of patients with systemic immune-based rheumatic diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The genes encoding full-length recombinant BLyS, APRIL, and FLAG-tagged BLyS were cloned into the mammalian expression vector pEE12.1 (Lonza Biologics, Slough, U.K.) using appropriate restriction sites. DNA prepared from each construct was cotransfected into HEK 293T cells by transient transfection using Lipofectamine (Invitrogen, San Diego, CA) and the cell culture supernatant harvested 3 days posttransfection.
ELISA
Immulon-2 plates (Dynex, Chantilly, VA) were coated by dispensing 100 µl of capture Ab solution (3 µg/ml in PBS) into individual wells followed by overnight incubation at 4°C. The next day the coating solution was removed, plates were washed two times with PBS, and blocked by incubation with 200 µl of blocking buffer (PBS, 3% BSA) for 2 h at room temperature.
For expression analysis, serial dilutions of cell supernatants were prepared in diluent buffer (PBS, 0.1% Tween 20, 0.1% BSA) and dispensed into individual wells of Ab-coated plates. The plates were incubated for 2 h at room temperature. Plates were washed four times with PBS Tween, 100 µl of the biotinylated detection Ab (0.25 µg/ml in diluent buffer) were added to each well and plates were incubated for 2 h at room temperature. After washing, streptavidin-peroxidase conjugate (Vector Laboratories, Burlingame, CA) was added (0.25 µg/ml in diluent), plates were incubated for 1 h, and after washing, the peroxidase reaction was developed with the TMB substrate kit (Sigma-Aldrich, St. Louis, MO). Light absorbance was measured at 450 nm using SpectraMax 3000 plate reader (Molecular Devices, Sunnyvale, CA) and dilution curves were generated in coordinates OD450 sample dilution.
For quantitative analysis of serum from normal donors and patients, a set of appropriate standards (BLyS, APRIL, or BLyS/APRIL heterotrimer) was prepared and processed along with serum samples in the same manner as described above.
mAbs against BLyS and APRIL
mAbs against BLyS and APRIL were generated using standard hybridoma technology. BALB/c mice were given a s.c. injection of 50 µg of the correspondent recombinant protein in CFA (Sigma-Aldrich), followed by three additional injections of 25 µg of the same protein in IFA at 2-wk intervals. Two weeks later, an i.p. injection of 25 µg of the protein in PBS was given, mice were sacrificed 4 days later, and splenocytes were harvested for fusion.
Splenocytes from one spleen were fused with 2 x 107 P3x63Ag8.653 plasmacytoma cells using PEG 1500 (Boehringer Mannheim, Indianapolis, IN), according to a manufacturer modification of an earlier described method (25). After fusion, the cells were resuspended in 400 ml of hypoxanthine/aminopterin/thymidine medium, containing 20% FBS, 4% of Hybridoma Supplement (Boehringer Mannheim), and distributed into 96-well plates for selection. Two weeks later, hybridoma supernatants were screened for specific Abs using solid-phase ELISA on Ag-coated plates. Top binders were identified by an ELISA modification of an affinity ranking procedure described elsewhere (26), and after two rounds of cloning by limiting dilutions, the corresponding hybridomas were expanded in ascites in syngenic mice. The Abs were affinity purified on a Protein A column (HighTrap; Amersham Pharmacia Biotech, Piscataway, NJ).
The B3-specific Ab was generated by phage display
technology using a human single-chain variable fragment (scFv) phage
display library (27). The lead scFv was isolated by
panning the library on immobilized BLyS. The binding specificity was
confirmed by phage ELISA on BLyS-coated plates as described elsewhere
(28). The VH and
VL genes from the scFv were converted to
full-length 
1 and 
formats using, correspondingly, the pEU1 and
pEU3 vectors (Cambridge Antibody Technology, Cambridge, U.K.)
according to a procedure described by Persic et al. (29).
The resulting constructs were expressed in HEK 293T cells and the Ab
purified from the culture media on a Protein A column.
Polyclonal Abs (pAbs) against BLyS and APRIL
Rabbits were given a s.c. injection of 100 µg of the BLyS or APRIL recombinant protein in CFA (Sigma-Aldrich), followed by several boosts of 50 µg in IFA at biweekly intervals. After the second boost, serum samples were analyzed for specific Ab titers by solid-phase ELISA on Ag-coated plates. After an additional boost, serum titers were high enough, serum was collected and pooled, and the Abs were purified by affinity chromatography using Affigel-10 columns (Bio-Rad, Hercules, CA) with immobilized BLyS or APRIL.
Immunoprecipitation and Western blotting analysis
Supernatants (500 µl/IP) from transiently transfected HEK293T cells were immunoprecipitated using 1 µg of the indicated Abs and 50 µl of protein A or protein G Sepharose for 2 h at 4°C. The beads were then washed three times with ice-cold wash buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 0.875% Brij 96, 0.125% Nonidet P-40, 1 mM Na3VO4, 5 mM NaF, 10 µg/ml each of leupeptin and aprotinin) and resuspended in Laemmli sample buffer. Proteins were separated on a 10–20% SDS-PAGE gradient gel, transferred to polyvinylidene fluoride membranes (Immobilon-P) and Western blotted using 1 µg/ml of the indicated pAb in 3% milk-PBS-T (PBS containing 0.2% Tween), followed by a secondary donkey anti-rabbit HRP-conjugated secondary Ab. Western blots were visualized by ECL (Pierce, Rockford, IL).
Human and murine B cell proliferation
Human tonsillar B cells were isolated by Ficoll centrifugation followed by negative selection using MACS magnetic beads (Miltenyi Biotec, Auburn, CA). Spleen cells were isolated from 6- to 10-wk-old female BALB/c mice by Ficoll centrifugation. B cell proliferation was assessed in the presence of Staphylococcus aureus cells (1/100,000 final dilution; Pansorbin; Calbiochem, La Jolla, CA) and protein concentrations ranging from 90 ng/ml to 0.01 pg/ml. Cells were resuspended at 1 x 105/well in a final volume of RPMI 10% FBS containing 1 x 10-5 M 2-ME, and incubated in the presence of the indicated factors for 72 h. The cells were then pulsed with 0.5 µCi/well of [H3]thymidine for another 20 h. Incorporation of thymidine was used as a measure of cellular proliferation.
For Ab or receptor (Fc fusions) inhibitions, cells were incubated in the presence of 3 ng/ml of either BLyS or APRIL/BLyS heterotrimer, and neutralizing activity was tested at concentrations ranging from 10 µg/ml to 100 pg/ml (six 10-fold dilutions).
Protein purification
Preparation of affinity columns. Three affinity columns with immobilized anti-BLyS homotrimer (B3), anti-APRIL homotrimer (A3), or anti-HT Ab were prepared in a similar way. A total of 10 mg of the Ab in 20 ml of PBS were mixed with 10 ml of preactivated AffiGel 10 (Bio-Rad) in 50 ml blue cup tube (Falcon; BD Labware, Franklin Lakes, NJ) and rotated overnight at 4°C. The gel was packed on the next day into a 10-ml column (Kimble/Kontes, Vineland, NJ), washed twice with 0.1 M Glycine-HCl (pH 2.5), and equilibrated with PBS.
Homotrimer absorption. Supernatants from transfected NSO cells were concentrated by tangential flow filtration on Pellicon XL (Millipore, Bedford, MA) using a membrane with the cut-off limit 10 kDa. The concentrated material (30–50 ml) was applied to an affinity column with immobilized anti-A3 Ab at a flow rate of 0.3 ml/min. The absorption cycle was repeated two more times. The column was regenerated between cycles by elution of the bound material with 0.1 M glycine-HCl (pH 2.5) and reequilibration with PBS. The same three absorption cycles were repeated on a column with immobilized anti-B3 Abs.
Affinity purification of heterotrimers. The flow-through after the last absorption cycle was then applied to a column with immobilized anti-HT Ab. The column was extensively washed with PBS and the bound BLyS/APRIL heterotrimer was eluted with 0.1 M Glycine-HCl (pH 2.5). The eluted material was immediately neutralized with PBS, aliquotted, and kept frozen at -80°C.
Patients and patient material
Patients admitted to the Los Angeles County and University of Southern California Medical Center and seen in consultation by the Rheumatology Service or patients receiving out-patient medical care at the Rheumatology Clinics of Los Angeles County and University of Southern California Medical Center (Los Angeles, CA) or the Edward R. Roybal Comprehensive Health Center (Los Angeles, CA) were recruited for this study. No exclusions were made on any basis other than an inability to give informed consent. Diagnoses were based on established clinical criteria (30). After obtaining written informed consent, venous blood was collected by venipuncture, permitted to clot, and stored at -70°C until shipment to Human Genome Sciences (Rockville, MD) on dry ice by overnight carrier for serologic evaluation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To address whether both APRIL and BLyS are
up-regulated in disease, we generated a panel of BLyS- and
APRIL-specific mAbs and pAbs to use as ELISA capture and detection
reagents, respectively. Samples collected from normal donors and from
patients with systemic immune-based rheumatic diseases were analyzed.
For BLyS, two ELISA formats were tested: two different mAbs to BLyS
were used as capture reagents, while the detection reagent was, in each
case, a pAb to BLyS. We found that while the two capture mAbs were able
to recognize recombinant BLyS in a similar fashion, as shown by their
standard curves (Fig. 1
A), the
mAb2 consistently detected higher levels of BLyS in both normal and
patient samples (Fig. 1B). We also analyzed the same samples
for APRIL expression and found that APRIL was slightly up-regulated in
patient samples compared with normal donors (Fig. 1C). The
fact that the mAb2 to BLyS detected higher levels of protein in both
sets of samples suggested two possibilities: 1) that this Ab
cross-reacted with APRIL, and was detecting both
B3 and A3; 2) that this Ab
was recognizing another form of BLyS, in addition to the described
homotrimeric molecule. The fact that APRIL and BLyS are secreted by the
same cell types in response to similar types of stimuli and that they
share the use of two receptors prompted us to ask whether they could
form heterotrimeric molecules, composed of one APRIL molecule and two
BLyS molecules (AB2) or two APRIL molecules and
one BLyS (A2B), in addition to the known
homotrimeric complexes. The discrepancy in the results observed between
the two ELISA formats could be explained if these heterotrimeric forms
of APRIL and BLyS did indeed exist and only mAb2 were able to
recognize them.
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To analyze whether APRIL and BLyS could form heterotrimeric
complexes (designated as HT, while BLyS and APRIL homotrimers are
designated as B3 and A3,
respectively), and to rule out that the differences observed with the
two mAbs were not due to cross-reactivity with APRIL, we first
transiently expressed full-length APRIL and BLyS cDNAs, alone or in
combination, in HEK293T cells. Supernatants from these cells were
collected and analyzed by immunoprecipitation and Western blotting
using a panel of Abs. We first immunoprecipitated the supernatants with
the two mAbs that we had previously characterized as able to bind
recombinant BLyS, and Western blotted the membrane with either a pAb to
BLyS or one to APRIL (Fig. 2). We found
that the first Ab (mAb1, designated as
anti-B3) was able to efficiently
immunoprecipitate homotrimeric BLyS (Fig. 2B, lane
2, third panel), but APRIL was not coimmunoprecipitated
when the two molecules were coexpressed (Fig. 2A, lane
2, fourth panel). In contrast, the second Ab (mAb2,
designated anti-HT) was able to immunoprecipitate a complex in
which both molecules were present (Fig. 2A, lane
4, fourth panel) in addition to immunoprecipitating the
B3. The fact that this Ab was not able to
immunoprecipitate APRIL when expressed in the absence of BLyS (Fig. 2A, lane 4, second panel) suggests
that its ability to detect higher levels of BLyS in human serum samples
was not due to cross-reactivity with APRIL or to nonspecific binding. A
similar analysis was performed by immunoprecipitating with an mAb to
APRIL (designated anti-A3) or with a pAb to
APRIL, followed by Western blotting analysis of the membranes with a
pAb to BLyS. The mAb anti-A3 only weakly
detected the heterotrimer, but specifically recognized the homotrimeric
APRIL molecule (Fig. 2, A and B, respectively,
lane 6, lower panels), while the pAb efficiently
isolated both the homotrimeric APRIL and the heterotrimeric APRIL/BLyS
molecules (Fig. 2, A and B, respectively,
lane 7, lower panels). Similarly, the polyclonal
anti-BlyS Ab was able to recognize both homotrimer BLyS and the
heterotrimer (data not shown). To rule out any nonspecific binding due
to the use of polyclonal serum as an immunoprecipitating reagent, we
performed a similar experiment using a FLAG-tagged form of BLyS (Fig. 2C). Immunoprecipitations with an anti-FLAG mAb followed
by Western blotting with the anti-APRIL pAb, resulted in a specific
coimmunoprecipitation of the FLAG-tagged form BLyS with APRIL, while no
association was observed with the nontagged form of BLyS (Fig. 2C, lanes 5 and 6, second
panel). In contrast, immunoprecipitating with the anti-BLyS
pAb resulted in coimunoprecipitation of both forms of BLyS with APRIL
(Fig. 2C, lanes 5 and 6,
first panel).
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, A and B), the
anti-A3/anti-APRIL pAb and the
anti-B3/anti-BLyS pAb formats
specifically recognized the homotrimeric form of APRIL and BLyS,
respectively. None of the other ELISA formats detected significant
levels of protein, indicating that there was no Ab cross-reactivity. In
contrast, supernatants collected from cells coexpressing APRIL and
BLyS, using the Ab that we had previously used to coimmunoprecipitate
the two molecules as a capture reagent (anti-HT) and the polyclonal
anti-APRIL Ab as a detector, we were able to detect significant
levels of heterotrimer (Fig. 3C). The polyclonal/pAb format
appeared to be the most sensitive in recognizing heterotrimeric
molecules (Fig. 3C, 
). The culture supernatant also
contained high levels of A3 and
B3 that were recognized by the
anti-A3 and anti-B3
capture mAbs, respectively.
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summarizes the binding
characteristics of the mAbs and pAbs described.
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Having characterized three Abs that were able to recognize
homotrimeric BLyS (anti-B3), homotrimeric
APRIL (anti-A3), and the heterotrimeric
molecules (anti-HT), respectively, we designed a purification
strategy that took advantage of these features. Supernatant generated
from a 200-ml culture of mouse myeloma NSO cells transiently
transfected with APRIL and BLyS was first depleted of the two types of
homotrimers (A3 and B3) by
affinity purification using an anti-A3 Ab
column, followed by an anti-B3 column. The
flow-through material was then analyzed by ELISA (using specific
anti-A3 or anti-B3
Abs) to confirm that the majority of homotrimeric BLyS and APRIL had
indeed been removed (data not shown). The BLyS/APRIL heterotrimeric
molecules were purified by absorbing the homotrimer-depleted material
on an Ab column made with the anti-HT Ab followed by acidic
elution. This material was analyzed by Western blot analysis and
quantitatively compared with known amounts of the recombinant APRIL and
BLyS protein standards (Fig. 4). The
heterotrimer standard appeared to be stable as the low levels of
A3 and B3 did not increase
over time. An ELISA analysis performed 6 mo after purification excluded
the spontaneous reformation of B3 and
A3(data not shown).
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). Although we have previously shown
that BLyS is an extremely potent B cell costimulator, capable of
inducing B cell proliferation at very low concentrations
(2), in our hands, APRIL weakly induces B cell
proliferation in the same range of concentrations (100 ng/ml to 0.2
pg/ml). However, the heterotrimer composed of APRIL and BLyS induced
human and murine B cell proliferation at concentrations similar to
those observed with recombinant BLyS (Fig. 5, A and
B). We then tested the set of mAbs that recognize either the
homotrimers or the heterotrimers for their ability to inhibit BLyS or
heterotrimer-induced human B cell proliferation.
Anti-B3 efficiently neutralized BLyS-induced
proliferation, but was not effective in inhibiting the activity induced
by the heterotrimer (compare Fig. 6, A and B). In contrast, the mAb recognizing both
homotrimer BLyS and the heterotrimers (anti-HT) was able to inhibit
proliferation induced by recombinant BLyS and by the purified
heterotrimers. The mAb against APRIL
(anti-A3) did not have any neutralizing
activity. These results confirm that the heterotrimer-induced B cell
proliferation is not due to the presence of any contaminating
homotrimeric BLyS.
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, C and D,
TACI-Fc efficiently neutralized BLyS and heterotrimer-induced
activation. BCMA-Fc and BAFFR-Fc significantly affected BLyS-induced
proliferation but did not efficiently inhibit the proliferation
observed in response to the heterotrimer. Similar results were obtained
when murine splenic B cells were used (data not shown). APRIL/BLyS heterotrimers are expressed in patients with systemic immune-based rheumatic diseases
Having shown that APRIL and BLyS can form active heterotrimers and
having characterized ELISA formats that would specifically recognize
this type of complex, we analyzed serum samples from 15 patients with
systemic immune-based rheumatic diseases and six normal controls. The
diagnoses of the patients included the following diseases: SLE, RA,
Reiter’s syndrome, psoriatic arthritis, polymyositis, and ankylosing
spondylitis. Two ELISA formats characterized by different capture Abs
were used—anti-HT mAb and anti-BLyS pAb—while the
anti-APRIL pAb was used as a detecting agent for both formats. In
addition to these ELISA formats, the patient serum samples were also
tested for A3 and B3 levels
(Table II). The average level of
BLyS/APRIL heterotrimer found in the majority of normal donors was
lower than 0.5 ng/ml. In the samples from the autoimmune patients, the
levels of APRIL/BLyS heterotrimer detected were significantly higher
(median was 3.11 ng/ml and 32.4 ng/ml using the anti-HT mAb or the
anti-BLyS pAb as capture reagents, respectively) than what was
detected in the normal controls. The levels of APRIL were also
significantly increased in rheumatic disease patients (median level 2.8
ng/ml) compared with normal donors, which suggests that the increase in
both APRIL and BLyS expression might contribute to the secretion of
heterotrimers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Shared receptor usage among the TNF ligands has been described for many
proteins (1). For example, LIGHT and LT both bind HveA
and LTR, but LT also shares two receptors with TNF (TNFR1 and
TNFR2). In general, all the members of this superfamily have been
characterized as homotrimeric molecules, with the exception of the
lymphotoxin-/lymphotoxin- heterotrimer. Although only
homotrimeric LT has been observed as a secreted active protein,
membrane-bound forms of the lymphotoxin-/lymphotoxin-
heterotrimers are known to bind different receptors and to elicit
different biological responses (33). The possibility that
shared receptor usage may actually reflect heterotrimerization of the
ligands has not been previously investigated.
The fact that BLyS and APRIL share significant protein homology, are secreted by the same type of cells in response to similar stimuli, and share two receptors prompted us to ask whether they could form biologically active heterotrimeric molecules.
In this paper we show that, when coexpressed, APRIL and BLyS can indeed form heterologous molecules. These, in principle, may be composed of either two APRIL molecules and one BLyS, or one APRIL and two BLyS molecules. Even though we do not have the tools to distinguish the two types of heterotrimers (A2B and AB2) from one another, Abs that specifically recognize the homotrimeric forms of BLyS and APRIL have allowed us to isolate and study the APRIL-BLyS complexes.
Using a human B cell proliferation assay, we were able to show that the heterotrimeric material we isolated from transfected HEK 293T cells was active. This heterotrimer-induced B cell proliferation could only be blocked by the mAb that recognizes heterotrimers in addition to B3. In contrast, an Ab that specifically neutralized B3-induced activation had no effect on BLyS/APRIL heterotrimer-induced proliferation. In addition, the Fc-fusion form of TACI efficiently inhibited both BLyS homotrimer- and heterotrimer-induced proliferation. The fact that BCMA-Fc and BAF-FR-Fc had a more modest effect raises more questions regarding the function of these receptors in the context of B cell regulation and survival. Recent reports have shown that BAFF-R may be the main receptor for BLyS; however, maturation-associated variations in BLyS receptor expression (34) suggest that TACI, BCMA, and BAFF-R may play different roles during the various stages of B cell maturation. The fact that heterotrimer-induced signals appear to be more efficiently mediated by TACI suggest that APRIL/BLyS heterotrimers may be active on mature B cells as TACI is expressed at higher levels on mature rather that immature B cells. Inasmuch as APRIL binds cells (adenocarcinoma tumor cells) that do not express either TACI or BCMA, and does not bind to BAFF-R, there likely is an as yet unidentified specific receptor for this ligand.
The Abs we have characterized have allowed us to show by ELISA that BLyS-APRIL heterotrimers are present in serum samples from patients with systemic immune-based rheumatic diseases, in some cases at very high levels. The polyclonal format consistently detected higher levels of heterotrimers in the patients compared with the anti-HT mAb capture format. This may be due to the fact that the polyclonal serum recognizes both forms of the heterotrimer (A2B and AB2) while the anti-HT mAb might preferentially bind to one form. In addition, the discrepancies between the two ELISA formats may be in part due to low avidity nonspecific interactions of the polyclonal serum. Larger numbers of samples will need to be analyzed to assess the frequency of patient samples in which heterotrimers can be observed in different diseases and how this correlates to the expression of B3 and A3. Elevated serum BLyS levels have been found in patients with RA, SLE, and Sjögren’s syndrome. Our findings suggest that both homotrimeric and heterotrimeric BLyS molecules may be deregulated in various rheumatic diseases; therefore, analysis of relative levels of homotrimeric and heterotrimeric molecules in samples from patients with different conditions will be of great interest.
The biological activity of the heterotrimeric molecules and their elevated presence in a subset of patients with systemic immune-based rheumatic diseases suggests that, for therapeutic purposes, an Ab or a decoy receptor recognizing the heterotrimeric forms of BLyS/APRIL may represent an intriguing alternative therapy in such a group of patients. Moreover, such a reagent may allow to distinguish patient subsets within an autoimmune disease.
In conclusion, we have shown that BLyS and APRIL can form biologically active heterotrimers that, similar to homotrimeric BLyS, appear to be deregulated in disease. Our findings further suggest that these heterotrimeric molecules may play a role in the pathology of various autoimmune diseases and also raise the possibility that other TNF ligands may form active soluble heterotrimers.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thi-Sau Migone, Department of Preclinical Development, Human Genome Sciences, 9410 Key West Avenue, Rockville, MD 20850. E-mail address: thi_migone{at}hgsi.com
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; scFv, single-chain variable fragment; pAb, polyclonal Ab; A3, APRIL homotrimer; B3, BLyS homotrimer.
Received for publication May 6, 2002. Accepted for publication August 2, 2002.
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 B. D. Hondowicz, S. T. Alexander, W. J. Quinn III, A. J. Pagan, M. H. Metzgar, M. P. Cancro, and J. Erikson The role of BLyS/BLyS receptors in anti-chromatin B cell regulation Int. Immunol., April 1, 2007; 19(4): 465 - 475. [Abstract] [Full Text] [PDF]
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P. K. A. Mongini, J. K. Inman, H. Han, R. J. Fattah, S. B. Abramson, and M. Attur APRIL and BAFF Promote Increased Viability of Replicating Human B2 Cells via Mechanism Involving Cyclooxygenase 2. J. Immunol., June 1, 2006; 176(11): 6736 - 6751. [Abstract] [Full Text] [PDF]
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D. A. Culton, B. P. O'Conner, K. L. Conway, R. Diz, J. Rutan, B. J. Vilen, and S. H. Clarke Early Preplasma Cells Define a Tolerance Checkpoint for Autoreactive B Cells J. Immunol., January 15, 2006; 176(2): 790 - 802. [Abstract] [Full Text] [PDF]
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A. L. Gavin, B. Duong, P. Skog, D. Ait-Azzouzene, D. R. Greaves, M. L. Scott, and D. Nemazee {Delta}BAFF, a Splice Isoform of BAFF, Opposes Full-Length BAFF Activity In Vivo in Transgenic Mouse Models J. Immunol., July 1, 2005; 175(1): 319 - 328. [Abstract] [Full Text] [PDF]
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J T Merrill BLyS antagonists and peptide tolerance induction Lupus, March 1, 2005; 14(3): 204 - 209. [Abstract] [PDF]
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P. Scapini, A. Carletto, B. Nardelli, F. Calzetti, V. Roschke, F. Merigo, N. Tamassia, S. Pieropan, D. Biasi, A. Sbarbati, et al. Proinflammatory mediators elicit secretion of the intracellular B-lymphocyte stimulator pool (BLyS) that is stored in activated neutrophils: implications for inflammatory diseases Blood, January 15, 2005; 105(2): 830 - 837. [Abstract] [Full Text] [PDF]
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W Stohl A therapeutic role for BLyS antagonists Lupus, May 1, 2004; 13(5): 317 - 322. [Abstract] [PDF]
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 E. Castigli, S. Scott, F. Dedeoglu, P. Bryce, H. Jabara, A. K. Bhan, E. Mizoguchi, and R. S. Geha Impaired IgA class switching in APRIL-deficient mice PNAS, March 16, 2004; 101(11): 3903 - 3908. [Abstract] [Full Text] [PDF]
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B. Huard, L. Arlettaz, C. Ambrose, V. Kindler, D. Mauri, E. Roosnek, J. Tschopp, P. Schneider, and L. E. French BAFF production by antigen-presenting cells provides T cell co-stimulation Int. Immunol., March 1, 2004; 16(3): 467 - 475. [Abstract] [Full Text] [PDF]
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 A. L Gavin, D. Ait-Azzouzene, C. F. Ware, and D. Nemazee {Delta}BAFF, an Alternate Splice Isoform That Regulates Receptor Binding and Biopresentation of the B Cell Survival Cytokine, BAFF J. Biol. Chem., October 3, 2003; 278(40): 38220 - 38228. [Abstract] [Full Text] [PDF]
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 L. Gorelik, K. Gilbride, M. Dobles, S. L. Kalled, D. Zandman, and M. L. Scott Normal B Cell Homeostasis Requires B Cell Activation Factor Production by Radiation-resistant Cells J. Exp. Med., September 15, 2003; 198(6): 937 - 945. [Abstract] [Full Text] [PDF]
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M. Pelletier, J. S. Thompson, F. Qian, S. A. Bixler, D. Gong, T. Cachero, K. Gilbride, E. Day, M. Zafari, C. Benjamin, et al. Comparison of Soluble Decoy IgG Fusion Proteins of BAFF-R and BCMA as Antagonists for BAFF J. Biol. Chem., August 29, 2003; 278(35): 33127 - 33133. [Abstract] [Full Text] [PDF]
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