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Alexion Pharmaceuticals, New Haven, CT 06511
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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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It has been suggested that naturally occurring soluble forms of both B7-1 and B7-2 may exist. For example, one study identified a soluble factor in the medium from primary porcine endothelial cells that mediates T cell activation (11). It was hypothesized that this soluble factor is porcine B7-2 based on the observations that its activity is CD28 dependent and that B7-1 mRNA is not detectable in these cells. An additional report demonstrated detectable levels of soluble human B7-1 in the synovial fluid of arthritic patients (12). However, it has not been determined whether these putative soluble forms of B7-1 and B7-2 are encoded from alternatively spliced messages or derived from full-length molecules on the cell surface through enzymatic cleavage or cell death.
Several alternate transcripts exist for B7-1 and B7-2. For example, multiple murine B7-2 alternatively spliced products have been identified (13). Additionally, an alternatively spliced form of murine B7-1 lacking the Ig C region-like extracellular domain has been described (14). Resolution of the B7-1 genomic organization in mouse and human indicates that both the transmembrane and cytoplasmic domains are encoded by separate exons, making it possible to exclude these regions without disrupting the extracellular domains (15, 16). Further analysis of the murine B7-1 genomic organization identified an additional exon that encodes an alternative cytoplasmic domain, confirming the presence of at least one downstream splice site (17). Taken together, these studies support the possibility that soluble B7 molecules could be generated through alternative splicing.
In this study, we describe the cloning of the porcine B7-1 homologue. Furthermore, this is the first study of an alternatively spliced form of B7-1 that encodes a soluble protein lacking both the transmembrane and cytoplasmic domains. Soluble porcine B7-1 (sB7-1)4 maintains its ability to bind both CTLA-4 and CD28 and can function to abrogate human T cell activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various mAbs reactive with porcine cell surface markers were purchased from VMRD (Pullman, WA), including anti-CD3 (clone 8E6), anti-IgM (clone PG145A), anti-class I (clone PT85A), and anti-class II (clone MSA3). The anti-porcine B7-2 mAb was generated at Alexion Pharmaceuticals (New Haven, CT). The functionally blocking anti-CD28 mAb 9.3 was a kind gift of Dr. Carl June (Department of Molecular and Cellular Engineering, University of Pennsylvania, Philadelphia, PA), and the anti-CD59 mAb MEM43 was obtained from Biodesign International (Kennebunkport, ME). Rabbit antiserum directed against porcine B7-1 was generated by repeated s.c. immunization with sB7-1 containing six histidine residues (sB7-1-His), according to methods routinely performed at Cocalico (Reamstown, PA). The IgG fractions from preimmune and immune sera were purified by passage over a protein A-Sepharose column (Pharmacia, Piscataway, NJ). The rabbit anti-histidine polyclonal IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-conjugated goat anti-rabbit and goat anti-mouse, and PE-labeled goat anti-rabbit secondary reagents were obtained from Zymed (South San Francisco, CA). Human CTLA-4Ig was purchased from Ancell (Bayport, MN), while recombinant porcine P-selectin-His was cloned and purified at Alexion Pharmaceuticals.
Cells and cell lines
To purify PBL, heparinized porcine peripheral blood was obtained from adult swine (Cocalico). Human peripheral blood was collected from healthy adult volunteers by venipuncture. Blood samples from either species were diluted 1/2 with HBSS (Life Technologies, Grand Island, NY), and the mononuclear fraction containing PBL was obtained by centrifugation over a Ficoll density gradient (Lymphocyte Separation Medium; Cellgro, Herndon, VA). Low density cells were collected from the interface and washed repeatedly in PBS containing 5% heat-inactivated bovine FCS (HyClone, Logan, UT). Viable cells were enumerated by trypan blue exclusion. The human T cell line, Jurkat, and the human B cell line, Raji, were obtained from American Type Culture Collection (Manassas, VA). Porcine aortic endothelial cells (PAEC) were purchased from Cell Systems (Kirkland, WA).
Cloning of porcine B7-1
Total RNA was prepared from freshly isolated porcine PBL using
the acid/guanidinium thiocyanate technique (18). Ten
micrograms of total RNA were heated at 65°C for 3 min, quenched on
ice, and subjected to first strand cDNA synthesis for 1 h at
37°C in the following 100 µl reaction mixture: 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 1.5 mM MgCl2, 10 mM
dithiothreitol, 0.20 mM of each dNTP, 0.5 µg
oligo(dT16), and 20 U of avian myeloblastosis
virus reverse transcriptase (Seikagaku, Rockville, MD). The following
oligonucleotide primers synthesized at Oligos Etc. (Wilsonville, OR)
were generated from regions of high homology between human and mouse
B7-1 sequence: 1) 5'-TGGCCCGAGTATAAGAACCGGAC-3' and 2)
5'-TCAGTTTCAGGATCTTGGGAAA-3'. A 5-µl aliquot of the cDNA pool was
used as a template in a 100 µl PCR reaction under the following
reaction conditions: 50 mM KCl, 10 mM Tris-HCl (pH 9), 1.5 mM
MgCl2, 0.1% (w/v) gelatin, 1% Triton X-100, 200
µM each dNTP, 2.5 U Taq DNA polymerase (Perkin-Elmer
Cetus, Norwalk, CT), and 25 pmol of each primer. PCR amplification was
performed for 30 cycles (94°C for 1 min, 50°C for 1 min, 72°C for
1 min), followed by a 1 cycle extension step at 72°C for 10 min. The
resulting 338-bp fragment was cloned into the pCR2.1 vector using the
T/A cloning system, as described by the manufacturer (Invitrogen, San
Diego, CA) and identified by sequence analysis as a B7-1 homologue. Two
gene-specific oligonucleotides were derived from the porcine B7-1
sequence, and a 250-bp fragment was generated by PCR. This DNA fragment
was used to screen a 
gt10 porcine macrophage library (a generous
gift from Dr. Michael Murtaugh, Department of Veterinary PathoBiology,
University of Minnesota, St. Paul, MN).
To screen the gt10 porcine macrophage library, approximately 1
x 106 phage were isolated on nitrocellulose
filters (Schleicher & Schuell, Keene, NH). Filters were denatured for
1.5 min (1.5 M NaCl and 0.5 N NaOH), neutralized for 5 min (1.5 M NaCl
and .05 M Tris, pH 8.5), rinsed in 3x SSC, air dried, and UV
cross-linked in a UV Stratalinker 2400 (Stratagene, La Jolla, CA).
Filters were then prehybridized in BSA/SDS buffer (1% BSA, 7% SDS,
0.5 M sodium phosphate buffer, pH 6.8, and 1 mM EDTA) for 2 h at
65°C before addition of the porcine B7-1 fragment, previously labeled
with 
-32P (NEN, Pittsburgh, PA) using the
Prime-It II random primer kit (Stratagene) to a sp. act. of 1 x
109 cpm/µg of DNA. Membranes were hybridized at
60°C overnight and subsequently washed using the following
conditions: two 30-min washes with 2x SSC/0.1% SDS at room
temperature, one 30-min wash with 0.5x SSC/0.1% SDS at 50°C, and
one 30-min wash with 0.2x SSC/0.1% SDS at 60°C. Positive plaques
present on duplicate lifts were purified and the B7-1 DNA was rescued
by PCR using primers that flanked the insertion site of the gt10
vector (Clontech, Palo Alto, CA). After cloning the PCR fragment into
pCR2.1-TOPO, both strands of the putative full-length clone were
sequenced using the chain termination method. Clones derived from
different PCR reactions were also sequenced to rule out potential
errors induced during amplification. The DNA templates were primed with
vector sequence primers flanking the multiple cloning site, or primers
constructed from internal cDNA sequence. All clones isolated from the
gt10 porcine macrophage library were identified as soluble porcine
B7-1 (sB7-1).
The transmembrane form of porcine B7-1 (tmB7-1) was isolated by RT-PCR of freshly isolated porcine lung RNA using an oligonucleotide from the 3' end of the sB7-1 coding region as the 5' primer (GCTACCAACACGATGCTTTCC) and oligo(dT16) as the 3' primer. Conditions for RNA isolation and RT-PCR were otherwise identical to those described above. The two major products resulting from the RT-PCR were cloned into pCR2.1-TOPO, and inserts were sequenced for identification.
Generation of His-tagged sB7-1
sB7-1 tagged with a carboxyl-terminal histidine hexapeptide (sB7-1-His) was generated in the mammalian expression vector Apex3P (19) by PCR amplification of B7-1 cDNA. The 5' primer (CCGGGGATCCCTTCTGTTTTCATCCTCATCAAGC) was derived from the 5' untranslated region (UTR) of B7-1 and contained a BamHI site for subcloning. The 3' primer (GGCCTGCAGGTCATCAATGGTGATGGTGATGGTGGCATTTTTGCCAGTTGAAGGTCTGTGAC) inserted the histidine tag just upstream of the stop codon and an Sse83371 subcloning site. For stable expression of sB7-1-His, 293-EBNA embryonic kidney cells (Invitrogen, Carlsbad, CA) were transfected with sB7-1-His/Apex3P, as previously described (19). Cells were grown in DMEM containing 5% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (D10 medium) with puromycin at a final concentration of 1 µg/ml. Selected cells were cloned by limiting dilution, and those producing high levels of protein were chosen by Western blot analysis of cell supernatants using rabbit anti-histidine polyclonal IgG. The sB7-1-His protein was purified by affinity chromatography using a nickel-charged nitrilotriacetic acid resin (Qiagen, Chatsworth, CA), as previously described (20). For transient transfections, the full-length untagged version of sB7-1 in pCR2.1-TOPO was subcloned into the mammalian expression vector Apex3P. sB7-1/Apex3P and sB7-1-His/Apex3P were then transfected into 293 cells, as described above. Cells were grown in D10 medium for 12 h and subsequently transferred into the serum-free medium HB PRO (Irvine Scientific, Santa Ana, CA) for 36 h. Cell supernatants were then collected for Western blot analysis.
Northern blot analysis
Total RNA from porcine alveolar macrophages was kindly provided
by Dr. Michael Murtaugh. Northern blot analysis was performed using the
NorthernMax Kit based on the manufacturer’s protocol (Ambion,
Arlington Heights, IL) and a total of 10 µg of RNA per lane. Blots
were hybridized with various
[-32P]UTP-labeled RNA probes that were
generated using the MAXIscript In Vitro Transcription Kit (Ambion).
RNA transcripts were synthesized from B7-1 DNA fragments contained in
pCR2.1-TOPO. These fragments consisted of either the complete 3' UTR of
sB7-1 (soluble probe), the complete transmembrane and cytoplasmic
regions of tmB7-1 (transmembrane probe), or the extracellular domain,
which is common to both the soluble and transmembrane forms of B7-1
(common probe). Levels of each transcript were determined by
densitometry using a Gel Doc 1000 (Bio-Rad, Hercules, CA) and NIH Image
1.61 software (downloaded from
http://rsb.info.nih.gov/nih-image/download.html).
PMA/ionomycin activation of porcine PBL
Porcine PBL intended for use as stimulator cells in mixed allogeneic or xenogeneic lymphocyte cultures (see below) were resuspended in R10 medium (RPMI containing 5 x 10-5 M 2-ME, 10 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% FCS) supplemented with 1 ng/ml PMA (Sigma, St. Louis, MO) and 400 ng/ml ionomycin (Sigma). The cells were seeded in 24-well plates at 2.5 x 106 cells/ml in a final volume of 2 ml, and incubated for 48–72 h at 37°C in 5% CO2 in air. Mitomycin C (50 µg/ml; Sigma) was added to the cells during the last 30 min of culture. Activated cells were then harvested and washed extensively to remove residual PMA/ionomycin and/or mitomycin C. Porcine PBL used in FACS analysis were stimulated with 20 ng/ml PMA and 200 ng/ml ionomycin for 48 h.
Immunofluorescence and flow cytometry
Activated cell populations were preincubated in PBS containing 5% goat serum. Cells were then incubated with purified IgG from hyperimmune serum from a rabbit immunized with sB7-1-His (rabbit anti-porcine-B7-1) or from preimmune rabbit serum, in combination with murine mAbs directed against porcine CD3, IgM, class II, and B7-2 cell surface Ags, in PBS containing 2% goat serum. Cells were washed in the same and then reacted with FITC-labeled goat anti-mouse Ig and with PE-labeled goat anti-rabbit Ig. The cells were again washed and analyzed for surface immunofluorescence using a FACSort flow cytometer and CellQuest Software (Becton Dickinson, Mountain View, CA). Further analysis was performed using WinMDI version 2.7 software (provided by Dr. Joseph Trotter, University of San Diego, CA).
In experiments performed to assess the binding of sB7-1 to the surface of Jurkat cells, sB7-1-His (2.5 µg/ml) was preincubated in HBSS containing either anti-CD28 mAb (25 µg/ml), anti-CD59 mAb (20 µg/ml), anti-CD3 mAb (20 µg/ml), human CTLA-4Ig (20 µg/ml), or buffer alone before addition to Jurkat cells (2.5 x 105 cells/reaction) for an additional incubation. Cells were then incubated with purified rabbit anti-porcine-B7-1 IgG (10 µg/ml), washed in HBSS, and finally incubated in the FITC-conjugated goat anti-rabbit secondary reagent (1/100 dilution). All incubations were performed for 30 min at 4°C. Jurkat/sB7-1-His binding was detected by cell surface immunofluorescence and flow cytometry, as described above.
Costimulation assays
The Jurkat T cell costimulatory assay has been described previously (21, 22). Briefly, PAEC were seeded in wells of 96-well microtiter plates at 5 x 104 cells/well in R10 medium, and cells were allowed to adhere overnight at 37°C. Jurkat T cells (1 x 106 cells/well) were then added to the wells in the presence or absence of 10 µg/ml PHA (Sigma) and either serial dilutions of sB7-1-His, 50 µg/ml of human CTLA-4Ig, or 100 µg/ml of porcine P-selectin-His. In some experiments, Raji cells (1 x 106 cells/well) were substituted for PAEC, but were added to the Jurkat cells at the initiation of the experiment. The cultures were maintained at 37°C in 5% CO2 for 24 h, at which time the culture supernatants were harvested. IL-2 was measured in supernatants using an ELISA kit (Quantikine Human IL-2 Immunoassay; R&D Systems, Minneapolis, MN), according to the manufacturer’s protocol. Briefly, serial dilutions of a human rIL-2 standard (R&D Systems) or culture supernatants were added, in duplicate, to ELISA wells that had been previously coated with a mAb specific for human IL-2, and plates were incubated overnight at 4°C. Unbound cytokine was removed by repeated washes. Bound IL-2 was detected using a second IL-2-specific Ab conjugated with HRP, followed by addition of a hydrogen peroxide/chromogen substrate. The reaction was stopped by the addition of 2 N sulfuric acid. The OD of each well was determined using a microtiter plate reader (Bio-Rad model 3550, Hercules, CA) set to 450 nm, with values corrected by subtraction of readings taken at 595 nm. IL-2 concentrations were calculated using Microplate Manager software (Bio-Rad).
Mixed lymphocyte reactions
Stimulator cells (mitomycin C-treated allogeneic human PBL or PMA/ionomycin-activated porcine PBL; 5 x 105 cells/well) and responder lymphocytes (human PBL; 5 x 105 cells/well) were combined in wells of a 96-well microtiter plate in the presence or absence of serially diluted sB7-1-His or of the indicated concentrations of CTLA-4Ig or porcine P-selectin-His (final volume, 0.2 ml/well). Cells were maintained in R10 medium for 4–5 days at 37°C in 5% CO2 in air. [3H]thymidine (1 µCi/well; NEN DuPont, Boston, MA) was added to the cell cultures during the last 16–18 h of the incubation. The cells were harvested onto glass fiber filters with an automated sample harvester (Wallac, Turku, Finland) and the filters were counted in a beta liquid scintillation counter (Wallac).
Western blot analysis
Supernatants from 293 cells transiently transfected with sB7-1/Apex3P, sB7-1-His/Apex3P, or Apex3P alone were subjected to SDS-PAGE (4–12% gradient gels) under reducing or nonreducing conditions. Proteins were transferred to nitrocellulose and the membrane was blocked for 1 h in blocking solution (Tris-buffered saline containing 5% dry milk). Blots were incubated for 1 h in fresh blocking solution containing either rabbit anti-histidine polyclonal IgG (0.2 mg/ml) or purified anti-porcine B7-1 polyclonal IgG (2 mg/ml). Blots were then washed three times with Ab wash solution (500 mM NaCl, 35 mM Tris, pH 7.4, 0.5 mM CaCl2, 0.1% SDS, 1% Nonidet P-40, and 0.5% deoxycholic acid) before the addition of fresh blocking solution containing HRP-conjugated goat anti-rabbit secondary Ab (1:5000; Zymed) for 15 min. Finally, blots were washed three times in Ab wash solution, incubated for 1 min in ECL Western blot reagent, and exposed to ECL Hyperfilm (both from Amersham, Arlington Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Signals generated by the interactions of CD28 and CTLA-4 with the B7 molecules have been shown to be critically involved in mediating experimental allograft rejection (23, 24). In addition, an important role for these molecules in cellular xenograft rejection is suggested by data showing enhanced graft survival when CD28-B7 interactions are inhibited (25). The demonstration of functional CD28-B7 interactions across the species barrier in the potentially clinically relevant porcine to human xenotransplant model could have significant therapeutic implications, given the demonstrated potency of the human anti-pig cellular immune response.
A recent study indicated that porcine B7-2 is recognized by human CD28,
and that this interaction promotes activation of human T cells by
porcine APCs (26). To further investigate the importance
of porcine B7 molecules in the human anti-pig immune response,
porcine B7-1 was cloned from a gt10 library generated from porcine
lung macrophages. In the initial cloning attempts, only cDNA encoding
sB7-1 was obtained from the macrophage library, at a frequency of
approximately one clone per 1 x 105 phage.
This full-length clone lacked both the transmembrane and cytoplasmic
domains. sB7-1 cDNA contained 1206 bp comprised of a 304-bp 5' UTR, a
215-bp 3' UTR including a processing/polyadenylation signal, and an
open reading frame that encoded 229 aa (data not shown; GenBank
accession number AF203442). The abnormally long 5' UTR observed for
sB7-1 corresponded to that of B7-1 reported for other species (4, 27). To obtain tmB7-1, RT-PCR was performed on porcine lung RNA
using a 5' primer generated from the end of the sB7-1 coding region and
oligo(dT16). A B7-1-specific band of
approximately 340 bp was generated that encoded a putative
transmembrane domain and a partial cytoplasmic domain, but lacked
sequence encoding the translational stop site and the 3' UTR (data not
shown; GenBank accession number AF203443). The truncation of tmB7-1
cDNA derived from reverse-transcribed porcine lung RNA and the lack of
detection of tmB7-1 in the oligo(dT)-primed porcine macrophage library
suggest strong 3' UTR secondary structure in this transcript.
The predicted amino acid sequences for sB7-1, tmB7-1, and human B7-1
(hB7-1) were compared (Fig. 1
A). Sequences were segregated
into domains based on exon boundaries identified for hB7-1
(15). Considering that sB7-1 and tmB7-1 amino acid
sequences were identical before the transmembrane domain, only sB7-1 is
depicted before this region. Excluding the transmembrane and
cytoplasmic domains, which are highly divergent between species,
porcine B7-1 and hB7-1 shared 65% sequence identity and an overall
conservation of the Ig V-like and Ig C-like structural domains
characteristic of other B7 molecules (28). The signal
peptide for sB7-1 was 29 aa in length, as determined by amino-terminal
sequencing of purified protein. Virtually all amino acid residues that
have been shown to be critical for the binding of B7-1 to both CD28 and
CTLA-4 (excluding methionine 47 and isoleucine 49) were highly
conserved (29, 30, 31, 32, 33). A clone containing the complete coding
region for tmB7-1 was not found, but based on sequence comparison with
various other species, the terminal amino acid is expected to be very
close to the translational stop site.
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B).
Sequence identity between the alternative forms of porcine B7-1 were
identical in exon 4, but showed no homology beginning with exon 5. A
stop codon was generated in the beginning of exon 5 for sB7-1. These
data suggest that sB7-1 is a splice variant lacking exons coding for
both the transmembrane and cytoplasmic domains and thus represent the
first report of a naturally occurring soluble form of B7-1. Northern analysis of porcine B7-1 splice variants
To determine the relative levels of expression of sB7-1
and tmB7-1 transcripts, Northern blot analysis was performed on total
RNA isolated from porcine alveolar macrophages. Reactive RNA species
were detected with either a sB7-1-specific probe, a tmB7-1-specific
probe, or a probe common to both sB7-1 and tmB7-1. The sB7-1-specific
probe hybridized with a single species of approximately 1.3 kb (Fig. 2). The size of this message approximates
the size of the sB7-1 cDNA clone isolated from the porcine macrophage
library. The tmB7-1-specific probe hybridized with a transcript of
approximately 3 kb, which represents the membrane form of porcine B7-1.
As expected, the tmB7-1-specific probe did not recognize the 1.3-kb
species. Finally, the common probe hybridized with both the 1.3- and
3-kb species. The relative densities of the two species reactive with
the common probe were determined using National Institutes of Health
Image 1.61 software. The tmB7-1 mRNA was present at a 3-fold excess
relative to sB7-1. These data indicate that both sB7-1 and tmB7-1
transcripts are well represented in porcine alveolar macrophages.
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Most studies have indicated that the B7-1 costimulatory molecule
is either undetectable or present on only a small subset of resting
human B and T cells, but that it is dramatically up-regulated in these
cell types upon stimulation by various means (34). To
assess the cell surface expression of porcine B7-1 on various subsets
of stimulated cells, porcine PBL were evaluated following stimulation
with PMA/ionomycin using two-color immunofluorescence (B7-1 was
detected by PE-, while other cell surface markers were detected by
FITC-conjugated secondary reagents). Following stimulation with
PMA/ionomycin, the majority of CD3-, class II-, and B7-2-positive cells
were also B7-1 positive (Fig. 3,
B, C and E, respectively). Virtually
all IgM-positive cells were B7-1 positive (Fig. 3D). These
data suggest that a membrane-bound form of porcine B7-1 is abundant on
the surface of both peripheral T cells and APC following cell
stimulation.
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The ligands for several porcine adhesion and costimulatory
molecules have been shown to be conserved across species, including
humans. These include the ligands for porcine E-selectin, VCAM, and
B7-2 (20, 26, 35). The fact that amino acids generally
shown to be critical for the binding of B7-1 to both CD28 and CTLA-4
are conserved in porcine B7-1 (Fig. 1A) suggests that this
molecule could interact with human CD28 and CTLA-4. To confirm this
prediction, purified sB7-1-His was incubated with human Jurkat cells.
Jurkat cells constitutively express CD28, but do not express CTLA-4
under any culture conditions (36). sB7-1-His specifically
bound to Jurkat cells (Fig. 4A), but failed to bind a
Jurkat derivative cell line that does not express CD28 (TIB 153, data
not shown). The binding of sB7-1-His to CD28 was effectively blocked by
an anti-CD28 mAb, further establishing the specificity of this
interaction. An isotype-matched irrelevant mAb did not interfere with
sB7-1-His/CD28 binding.
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B). By contrast, an isotype-matched irrelevant mAb had no
effect on sB7-1-His/CD28 binding. The interaction between porcine B7-1
and human CTLA-4 was also confirmed by ELISA, as plate-coated sB7-1-His
bound to CTLA-4Ig in a titratable fashion (data not shown). These data
demonstrate that a naturally occurring soluble form of porcine B7-1 has
the ability to bind both human CD28 and CTLA-4. Furthermore, the
binding of porcine B7-1 to human receptors suggests that this
interaction could play a role in T cell activation during pig to human
xenotransplantation. sB7-1 inhibits IL-2 production following costimulation of human Jurkat T cells
To examine whether sB7-1 could interact functionally with human T cells, sB7-1-His was titrated into the Jurkat costimulation assay and its effect on T cell activation was evaluated by detection of IL-2 in the culture supernatants. Jurkat cells do not constitutively elaborate IL-2, even in the presence of PHA, which provides a primary signal through the TCR. However, PHA signaling in the presence of APCs or a stimulatory anti-CD28 mAb results in significant IL-2 production (37).
Jurkat cells were incubated alone or were cocultured with PAEC or Raji
cells in the presence or absence of sB7-1-His, an irrelevant
histidine-tagged protein or human CTLA-4Ig. As expected, in the absence
of PAEC or Raji cells, Jurkat cells did not generate detectable IL-2
under any of the culture conditions tested (data not
shown). By contrast, Jurkat cells
generated high levels of IL-2 when stimulated with PHA in the presence
of either PAEC (Fig. 5A) or Raji cells (Fig. 5B).
Addition of sB7-1-His inhibited the production of IL-2 in a
dose-dependent manner, with maximal inhibition equivalent to that
observed by the addition of human CTLA-4Ig. Inhibition of IL-2
production was virtually complete at high doses of sB7-1-His,
regardless of the source of APCs (porcine or human) used to provide the
secondary signal. Addition of recombinant porcine P-selectin-His did
not significantly influence IL-2 production in these assays.
Nonspecific toxicity of the sB7-1-His preparation due to excipient
effects was excluded by evaluating similar volumes of dialysis buffer,
collected during the final dialysis of the protein (data not shown).
Since Jurkat cells do not express CTLA-4, these data suggest that
sB7-1-His inhibits IL-2 production by binding to CD28 and blocking
signaling through this molecule.
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To determine the potential effect of sB7-1-His on T cell
activation under conditions in which both CD28 and CTLA-4 molecules
were present, MLRs were performed. Short-term primary human allogeneic
and xenogeneic MLRs were established by coculturing human PBL with
mitomycin C-treated human (Fig. 6A) or porcine (Fig. 6B) PBL, respectively. MLRs were performed in the presence
of increasing amounts of sB7-1-His, an irrelevant histidine-tagged
protein, or CTLA-4Ig, and responder cell proliferation was assessed on
day 4–5 of culture. The addition of sB7-1-His effectively inhibited
human T cell proliferation in a dose-dependent fashion in response to
both allogeneic and xenogeneic stimulator cells (Fig. 6, A
and B, respectively). Addition of human CTLA-4Ig at
concentrations of 100 µg/ml also effectively inhibited cell
proliferation in both assays, while addition of porcine P-selectin-His
at 100 µg/ml had no effect. These results indicate that binding of
porcine sB7-1-His to CD28 and/or CTLA-4 ligands on human T cells
inhibits their activation by allogeneic or xenogeneic stimulation in a
concentration-dependent manner.
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The generation of sB7-1 through alternative splicing creates a
cysteine residue before a translational termination signal (Fig. 1).
Although this cysteine residue is included in the recombinantly
produced protein, the addition of a histidine tag on the carboxyl
terminus of sB7-1 could affect potential disulfide bond formation, and
thus dimerization of the molecule. To investigate the ability of the
native molecule to dimerize, sB7-1 was transiently expressed in 293
cells and compared with sB7-1-His under reducing and nonreducing gel
electrophoresis, followed by Western blot analysis. Under reducing
conditions, both proteins migrated as a doublet at approximately 40
kDa, with the histidine-tagged version of the protein running just
above the untagged version, most likely due to the hexapeptide tag
(Fig. 7B, lanes 2
and 3, respectively). Conversely, while sB7-1-His ran
identically under nonreducing conditions, the untagged version of sB7-1
showed an additional major species at approximately 80 kDa, presumably
due to dimerization of the molecule through cysteine bonding (Fig. 7C, lanes 2 and 3, respectively). As
expected, Ab reactive against the histidine tag recognized sB7-1-His,
but not the untagged version of the protein (Fig. 7A,
lanes 2 and 3, respectively). No reactivity was
observed in samples prepared from 293 cells transfected with vector
alone (Fig. 7, all panels, lane 1). These results suggest
that native sB7-1 may exist as both a monomer and dimer. Furthermore,
preliminary studies with sB7-1 preparations containing primarily
homodimer indicate that this multimer also functions to block T cell
activation in an MLR (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The naturally occurring soluble form of B7-1 described in the present study represents a potential regulatory component of the immune system that heretofore has not been described. A histidine-tagged version of sB7-1 effectively bound CD28 and CTLA-4 molecules and blocked T cell activation in both allogeneic and xenogeneic MLRs. B7-1 has previously been expressed as a recombinant soluble molecule by replacing the transmembrane and cytoplasmic domains with the Fc region of an Ab or with an oligo-histidine tag (2, 42, 43). These recombinant soluble B7-1 molecules (rsB7-1) also maintain their ability to bind both CD28 and CTLA-4 (2, 42, 43, 44). In addition, plate-immobilized rsB7-1 effectively stimulates T cells in conjunction with a primary signal (anti-CD3 mAb) (2, 43). The ability of these recombinant molecules to block T cell activation was not tested.
Inhibition of T cell activation by sB7-1-His most likely occurs through the inhibition of CD28 binding to B7-1 and/or B7-2 on the APCs. Since it has been shown that Ag presentation in the absence of costimulation through CD28 results in T cell anergy (5, 6), it is interesting to speculate that endogenous sB7-1 may exert this same effect in vivo by blocking the binding of CD28 to its ligands. Blockade of CD28 interactions with the B7 proteins may also reduce the production of Bcl-xL, a molecule important in preventing apoptosis, as engagement of CD28 has been shown to promote the production of this survival factor (45).
In the present study, the inclusion of a carboxyl-terminal histidine hexapeptide prevented B7-1 disulfide-linked dimer formation, as an untagged version of the molecule migrated primarily as a dimer under nonreducing electrophoretic conditions, while the histidine-tagged version ran entirely as a monomer. Apparently, the highly charged histidine tag restricts disulfide bond formation between cysteine residues found at the carboxyl end of the molecule. This finding raises the question of whether sB7-1-His may be functionally different from the untagged version of the molecule. Our preliminary studies with sB7-1 preparations containing primarily homodimer indicate that the homodimer also blocks T cell activation in an MLR (data not shown). In addition, the recent resolution of the crystal structure of human rsB7-1 indicates that this molecule undergoes a rapid monomer-dimer exchange that favors nondisulfide-bonded homodimer formation (46). These data suggest the possibility that sB7-1-His utilized in the present study may also exist as a homodimer.
The ability of sB7-1 to form a stable homodimer may have other important functional consequences. Based on crystal structure data, it has been predicted that the increase in avidity between B7-1 and CTLA-4 homodimers may serve to stabilize the B7-1/CTLA-4 signaling complex, which would facilitate the termination of T cell activation (46). It is interesting to speculate that cysteine bond formation between sB7-1 molecules would further favor the generation of stable homodimers. However, it is not known whether a sB7-1 homodimer would activally signal through CTLA-4 or alternatively, block CTLA-4 signaling by competing with B7-1 on the surface of the APC. Definitive studies addressing the physiologic function(s) of sB7-1 are currently underway.
Alternative splicing of a heteronuclear transcript can result in the generation of a soluble molecule from an otherwise membrane-linked protein. This generally occurs by removal of an exon coding for the transmembrane domain by use of an alternative downstream splice site. Examples of soluble proteins that are generated through this mechanism include LFA-3, P-selectin, and many of the cytokine receptors (38, 47). Alternatively spliced products have also been described for both B7-1 and B7-2 (13, 14), but to our knowledge, this is the first report of an abundant endogenous mRNA that lacks the transmembrane and cytoplasmic domains and encodes a functional, soluble B7 protein.
Evidence that sB7-1 reported in the present study is indeed an alternatively spliced product and not an incompletely processed mRNA with the stop codon generated from unspliced intron sequence includes the following: 1) the 1.2-kb full-length cDNA for sB7-1 corresponds in size to one of two species identified by Northern blot analysis of RNA derived from porcine macrophages, and a probe generated from sequence specific to sB7-1 (3' UTR sequence) recognized this transcript; 2) a known processing/polyadenylation signal is found at the end of the 3' UTR sequence of sB7-1; 3) the sB7-1 message is well represented in the porcine macrophage cDNA library; and 4) there is an absence of a splice donor site at the point that the sB7-1 cDNA sequence diverges from that of tmB7-1. In addition, using PCR analysis of porcine genomic DNA, we demonstrated that the end of the sB7-1 coding region is not juxtaposed to the downstream untranslated region, providing further support that this sequence is not unspliced intron (data not shown).
A tmB7-1-specific probe recognized a mRNA species of approximately 3 kb by Northern blot analysis, which corresponds to that reported previously for stimulated porcine lymph node cells (11). However, this study did not demonstrate the presence of the sB7-1 transcript in lymph node cell RNA, which may indicate its absence or low level of expression in this tissue source. Differential expression of alternatively spliced B7 products in various cell types and in response to different stimuli has been previously reported (13, 48).
Although mRNA for sB7-1 was not as abundant as the tmB7-1 transcript (3-fold less), it was well represented in the alveolar macrophage library. The complete absence of tmB7-1 cDNAs allowed the total number of clones representing sB7-1 to be determined. Of a total of approximately 3 x 106 plated phage, approximately 30 sB7-1 clones were isolated (1 clone per 1 x 105 phage). This frequency is indicative of a low-abundance mRNA (mRNAs occurring at less than 14 copies per cell) with an average occurrence in an oligo(dT) reverse-transcribed library (49).
Recently, a soluble form of hB7-1 has been identified in the synovial fluid of arthritic patients (12). It is not known whether this soluble molecule is also encoded by an alternatively spliced message or rather, represents a result of enzymatic cleavage from the cell surface. Northern blot analysis has identified at least four different hB7-1 transcripts that may represent alternatively spliced molecules (4). Using RT-PCR, we have also cloned a putative alternatively spliced form of hB7-1 that encodes a protein lacking the transmembrane and cytoplasmic domains (data not shown). However, this species was not detectable by Northern blot analysis, suggesting either that it was not present in the source of RNA analyzed or it represented a rare transcript. The relevance of this alternatively spliced human soluble B7-1 is currently under investigation.
Based on anatomical and physiological considerations, the pig is considered the most likely candidate as a xenogeneic organ donor for human transplants. However, cellular mechanisms of rejection of pig organs mediated by recognition of porcine adhesion and/or costimulatory molecules are likely to represent at least one major barrier to the success of such transplants (50). For example, porcine E-selectin, VCAM, and B7-2 have all been shown to interact with the human homologues of their receptors and to mediate human leukocyte adhesion or T cell costimulation (20, 26, 35). In the present study, we show that porcine B7-1 is recognized by the human receptors CD28 and CTLA-4 and that it can also serve as a ligand for human T cell costimulation by porcine APCs. These data suggest that porcine B7-1 may represent a good candidate for blockade or targeted gene knockout in the xenotransplantation setting. However, the functional redundancy described for B7-1 and B7-2 suggests that blockade of B7-1 alone may not be sufficient to adequately inhibit T cell activation. Indeed, the combination of functionally blocking anti-murine B7-1 and B7-2 mAbs is required to effectively inhibit primary MLRs and allogeneic transplant rejection, while inhibition with anti-B7-1 mAbs alone does not (24, 51, 52). Thus, we are currently investigating the effectiveness of functionally blocking mAbs to porcine B7-1 and B7-2 either independently or in combination during xenotransplantation.
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Footnotes |
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2 Current address: Immunology, Cancer and Infectious Disease, Central Research Division, Pfizer Inc., Eastern Point Road, Groton, CT 06340.
3 Address correspondence and reprint requests to Dr. Russell P. Rother, Alexion Pharmaceuticals, Molecular Development, 25 Science Park, Box 15, New Haven, CT 06511.
4 Abbreviations used in this paper: sB7-1, soluble B7-1; hB7-1, human B7-1; PAEC, porcine aortic endothelial cell; tmB7-1, transmembrane B7-1; UTR, untranslated region.
Received for publication September 20, 1999. Accepted for publication March 28, 2000.
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), P-selectin-His (
), or
sB7-1-His (
). The cell cultures were maintained at 37°C for
24 h, at which time the supernatants were harvested and assayed by
indirect sandwich ELISA to determine the concentration of hIL-2, as
described in Materials and Methods. Results are
expressed as the mean of duplicate ELISA determinations, converted to
pg/ml IL-2 using Microplate Manager software.
), or P-selectin-His (
) or human or porcine
stimulator cells alone (