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INTRODUCTION |
CD8 has been shown to function in mediating signal transduction
and adhesion on a subset of cells within the T cell compartment and is
critically involved in the development of T cells expressing MHC class
I-restricted T cell receptor
(TCR)1 (1). CD8 is encoded by
two distinct genes, termed CD8
(or Lyt 2) and CD8
(or Lyt 3) and
expressed on the cell surface as a mixture of disulfide-linked
CD8 homodimers and CD8 heterodimers (2-7). CD8 is a
34-37-kDa transmembrane glycoprotein whose extracellular segment
contains a compact 122-amino acid (aa) N-terminal Ig-like domain and an
extended 48-residue stalk region. A 28-aa cytoplasmic domain, including
a cysteine motif responsible for interaction with p56lck,
follows a canonical hydrophobic transmembrane anchor. CD8 is a
32-kDa glycoprotein sharing a similar architecture as CD8 but with
<20% sequence identity (4, 5). The stalk regions of the CD8 and
chains are quite different in length, with the CD8 stalk being
10-13 residues shorter than that of CD8. Interestingly, the sialic
acid content of O-linked glycans adducted to CD8
selectively decreases on thymocytes and activated T cells compared with
that found on resting T cells. For its cell surface expression, CD8 requires association with the CD8 subunit, forming a CD8
heterodimer (6, 8). Moreover, CD8 genes are selectively expressed.
Although CD8 heterodimers are predominantly found on the surface
of TCR T cells and thymocytes, CD8 homodimers are
additionally expressed on a subset of 
T cells, intestinal
intraepithelial lymphocytes and natural killer cells (9, 10). Hence, it
is safe to conclude that the two sets of CD8 co-receptors subserve
distinct functions.
The importance of CD8 in p56lck-linked T cell activation and
signaling has been defined by multiple studies (11, 12). Although unable to bind p56lck directly, other findings emphasize the
contribution of CD8 to the efficacy of T cell recognition and its
ability to broaden the range of antigen recognition (13, 14). Several
lines of evidence including our own show that CD8 is a more
effective co-receptor than CD8 in enhancing the sensitivity to
peptide antigens as well as alloantigens recognized by TCRs (15, 16). A
role for the cytoplasmic portion of the CD8 chain in enhancing Lck
kinase activity and promoting T cell development has been suggested
(17, 18). In addition, at least for certain TCRs, the contribution of
CD8 as a co-receptor may be due, in large part, to its
extracellular components (16).
Monoclonal antibody blocking studies, cellular adhesion assays, and
direct molecular interaction studies as well as the recent studies of
the crystal structures of the Ig domain of the human CD8
(hCD8) dimer in complex with HLA-A2 and murine CD8
(mCD8) in complex with H-2Kb have shown that the
natural ligand of CD8 is the MHC class I molecule (19, 20). These
structures show unequivocally that one CD8 dimer binds to one
pMHC complex. Moreover, these findings are consistent with mutational
analyses indicating that the 3 loop of the MHC class I (MHCI)
molecule is the major CD8 binding component. Although the CD8
heterodimer is thought to bind to the MHCI 3 region as well,
currently little is known about the specific molecular details.
Given the importance of understanding the molecular interactions
between the CD8 and MHC molecules, recombinant CD8 ectodomain fragments
have been produced in a variety of systems (21-28) but without
successful secretion of homogeneous products. To overcome this
limitation, we expressed the Ig-like domain of mCD8 or the larger
ectodomain fragment utilizing an engineered leucine zipper (LZ) system
(29), which we previously applied to the expression of heterodimeric
soluble TCR and chains. The CD8 Ig-like domain protein
purified from Lec3.2.8.1 cells could be readily crystallized (20) in
complex with the murine MHC class I H-2Kb molecule loaded
with VSV8 octapeptide (30). The same strategy was used to produce the
Ig-like domain of soluble recombinant murine CD8 protein as well
as the entire extracellular segment. The binding of proteins to pMHC
was examined by surface plasmon resonance as well as by functional
inhibition studies of killing activity using N15 TCR (29) transgenic
(tg) cytolytic T cells. As detailed below, we offer an explanation for
the paradox as to why the transmembrane CD8 versus
CD8 co-receptors have such large differences in efficiency of
facilitating TCR recognition (15, 16) and yet bind to class I MHC with
comparable affinities.
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EXPERIMENTAL PROCEDURES |
Molecular Design and Engineering of Secreted CD8 Co-receptors
Using a Heterodimeric Coiled Coil Sequence--
To promote the
secretion of recombinant dimeric proteins in eukaryotic cells, the
N-terminal extracellular segment of the mCD8 chain (residues 1-122)
corresponding to the predicted Ig-like domain was fused via a flexible
linker (aa residues 123-137) and a thrombin cleavage site (aa residues
129-132) to either Acid-p1 or Base-p1 leucine zipper fragments (31)
forming CD8-A and CD8-B, respectively (Fig. 1A). For
this purpose, a 5'-primer encoding aa 1-5 of CD8 and a 3'-primer
encoding aa 115-122 plus 10 amino acids of flexible linker were used
for polymerase chain reaction of the CD8 Ig-like domain from the
pHbAPr-1 neo/Lyt2 cDNA clone (3). The resulting DNA fragment was
cloned into the pCR2 vector and sequenced. Subsequently, the error-free
CD8-Ig-like DNA fragment was then digested with SpeI and
BamHI and ligated with a BamHI-SpeI
DNA fragment encoding Acid-p1 or Base-p1 peptides to form the CD8-A
and CD8-B, respectively. The DNA fragments were then subcloned into
the XbaI site of the pEE14 vector (CellTech Ltd, Berkshire,
UK) to form plasmids, pEE14 CD8-A, and pEE14 CD8-B, respectively.
The CD8 construct pEE14 CD8-A was generated in a similar fashion
using a 5'-primer encoding residues 1-6 of murine CD8, a 3'-primer
encoding CD8 aa 109-115 and 10 amino acids of flexible linker and
the CD8 Ig-like domain from pHbAPr-1 neo/Lyt3 cDNA clone (8) as
a template. The error-free CD8 Ig-like DNA fragment then was
digested with XbaI and BamHI and ligated with the
BamHI-EcoRI DNA fragment encoding Acid-p1 to form
the CD8-A. Subsequently, the DNA fragment was subcloned into the
XbaI and EcoRI site of pEE14 vector to form
plasmid, pEE14 CD8-A. After sequence verification, the pEE14
CD8-A plus pEE14 CD8-B cDNAs or pEE14 CD8-B plus pEE14
CD8-A cDNAs were pairwise co-transfected into Lec3.2.8.1 cells
(32) to produce murine CD8-LZ or CD8-LZ proteins.
To avoid cysteine mispairing and incorrect disulfide bond formation, a
construct encoding the soluble full-length extracellular domain of
CD8 was terminated prior to cysteine residue 166. Moreover,
cysteine 151 in the stalk region was mutated to a serine residue. A 5'
oligonucleotide primer corresponded to aa 1-5 of CD8 and a
3'-primer encoding aa 148-165 and 10 amino acids of flexible linker
were used to polymerase chain reaction the CD8f from the
pHbAPr-1 neo/Lyt2 cDNA clone template. The DNA fragments generated
were cloned into the pCR2 vector, and sequenced. The error-free
CD8f DNA fragment then was digested with SpeI
and BamHI and ligated to the
BamHI-SpeI DNA fragment encoding Acid-p1 or
Base-p1 segments to form CD8f-A and
CD8f-B, respectively. The DNA fragments were then
subcloned into the XbaI site of pEE14 vector to generate the
pEE14 CD8f-A and pEE14 CD8f-B plasmids. The construction of pEE14 CD8f-A was generated in a
similar fashion to pEE14 CD8f-A, terminating immediately
prior to cysteine 150. As with CD8, the cysteine at aa 137 in the
murine CD8 stalk region was mutated to serine. The DNA fragment of
the CD8 full-length extracellular domain was generated by polymerase
chain reaction from the pHbAPr-1 neo/Lyt3 cDNA clone as a template
using the 5'-primer encoding aa 1-6 of CD8 and the 3'-primer
encoding aa 135-149 as well as 10 aa of flexible linker. The
error-free CD8f-A DNA fragment then was digested with
XbaI and BamHI and ligated to the
BamHI-EcoRI DNA fragment encoding the Acid-p1 to
form the CD8f-A. Subsequently, the DNAs were subcloned
into the XbaI and EcoRI sites of pEE14 vector to
form the plasmid pEE14 CD8f-A. After sequence
verification, the pEE14 CD8f-A plus pEE14
CD8f-B cDNAs or pEE14 CD8f-B plus
pEE14 CD8f-A cDNAs were pairwise co-transfected into
Lec3.2.8.1 cells to produce soluble murine CD8f-LZ or CD8f-LZ protein.
Expression of CD8 Ectodomain Fragments Using a Glutamine
Synthetase Vector--
To produce large quantities of soluble
recombinant murine CD8-LZ protein in Lec3.2.8.1 cells, a method
similar to that described in detail for soluble recombinant TCRs (33)
was employed. 20 µg of SalI linearized plasmid DNA pEE14
CD8-A and pEE14 CD8-B were used for transfection by a calcium
phosphate precipitation method using a Transfection MBS kit
(Stratagene) following the manufacturer's protocol. 48 h after
transfection, the cells were trypsinized, resuspended into 10 ml of
Glasgow minimal essential medium-supplemented containing 25 µM methionine sulfoximine and cultured onto 96-well
plates. Three to four weeks later, the growing clones were assayed for
secretion of soluble CD8 homodimer by ELISA. In brief, 10 µg/ml
of the anti-leucine zipper antibody, 2H11, was coated onto Immulon
plate (Dynatech) at room temperature for 2 h, and then the plates
were blocked with 1% bovine serum albumin in borate-buffered saline at
room temperature for 2 h. 50-µl culture supernatants of
individual clone were plated overnight at 4 °C, mixed with 5 µg/ml
of biotinylated anti-CD8 mAb 53.6.72 (34) for 2 h, and
developed with horseradish peroxidase-conjugated streptavidin (Sigma).
The positive clones were picked and transferred to 24-well plates.
Subsequently, the highest secretors were ranked by rescreening the
supernatants using an indirect capture with the anti-velcro mAb 2H11
(29) on BIAcore (Pharmacia Biosensor) (33). The identical ELISA was
used for detection of CD8f-LZ proteins. The
productions of murine CD8-LZ and CD8f-LZ were generated similarly using two pairs of SalI linearized
plasmid DNAs, pEE14 CD8-A/pEE14 CD8-B and pEE14
CD8f-A/pEE14 CD8f-B, respectively, for
transfection into Lec 3.2.8.1 cells. The stable clones producing
CD8-LZ or CD8f-LZ were identified by the ELISA
method described above except using 5 µg/ml of biotinylated anti-CD8 mAb YTS156 (35) as the detecting antibody. Transfections of
pEE14 CD8f-A/pEE14 CD8f-B and pEE14
CD8f-A/pEE14 CD8f-B were performed in CHO
cells as well.
Large Scale Production and Purification of CD8 Protein--
The
transfected Lec3.2.8.1 cell lines producing recombinant soluble CD8
(clones CD8-22-1 for CD8-LZ, CD8-21-7 for
CD8f-LZ, CD8-213-16 for CD8-LZ, and CD8-223-34
for CD8f-LZ) and CHO cells producing recombinant
soluble CD8 (clone 15 for CD8f-LZ and clone 21 for
CD8f-LZ) were cultured in glutamine-free Glasgow minimal essential medium-supplemented containing 10% dialyzed fetal
calf serum and 25 µM methionine sulfoximine and expanded for large scale protein production as described previously (33). The
Lec3.2.8.1 supernatants containing the CD8-LZ fusion proteins were filtered (Corning, 0.22 µM) and immunoaffinity
purified using the anti-leucine zipper mAb 2H11 according to an earlier
protocol (33). The bound CD8-LZ was eluted with low pH buffer (20 mM Tris, 50 mM citrate, 0.5 M NaCl,
10% glycerol, pH 4.0, adjusted with NH4OH) and immediately
adjusted to pH 7.0, using 1 M Tris-HCl, pH 9.5. CD8-LZ, CD8f-LZ, CD8f-LZ,
CHO-CD8f-LZ, and CHO-CD8f-LZ were
purified following a similar procedure. CD8 proteins that were used for
cytotoxicity experiments were affinity purified, concentrated to
35-100 mg/ml using a Centricon-10 concentrator (Amicon), and buffer
exchanged into PBS, pH 7.2. CD8 proteins that were employed for BIAcore
studies were concentrated, sized on a 1.6 × 60 cm Superdex 75 gel
filtration column (Amersham Pharmacia Biotech) to remove any
aggregates, and then concentrated and equilibrated in PBS. No CD8
aggregates were detected on reanalysis of the gel filtration-sized
proteins. The protein concentration of CD8 samples was determined using
a Bicinchoninic acid protein assay (Pierce) with bovine serum albumin standards.
BIAcore Studies--
All binding studies were performed with
PBS/Tween 20 (0.005%) on a BIA1000 surface plasmon resonance biosensor
(BIAcore Inc.). To study the CD8-H-2Kb interaction, we took
advantage of the CD8 leucine zipper constructs by capturing the
CD8-leucine zipper molecules with an anti-leucine zipper mAb coupled to
the sensor chip by standard
N-hydroxysuccinimide/N-ethyl-(dimethylaminopropyl) carbodiimide chemistry following standard procedures (BIAcore). This
approach aligns all CD8 molecules in a similarly ordered manner, making
them accessible for the H-2Kb interaction. Because 2H11 has
a relatively fast dissociation rate, we employed an additional mAb,
termed 13A12, generated in our laboratory (data not shown). 20 µl of
13A12 at 100 µg/ml in 10 mM NaAc, pH 4.5, were
immobilized on a CM5 sensor chip at a flow rate of 5 µl/min resulting
in ~5000 RU coupled. CD8f-LZ and H-2Kb
samples were buffer exchanged into PBS, which is equivalent to the
buffer in the reservoir. 20 µl of CD8f-LZ (1 µM) were injected onto the 13A12 surface at a flow rate
of 10 µl/min. After a 5-min dissociation period, 5 µl/min of
H-2Kb-VSV8 (64 µM) were injected onto the
13A12-CD8f-LZ surface. This procedure was repeated
using H-2Kb-VSV8 concentrations of 1-32 µM.
The specificity of the binding was assured by H-2Kb
injections (1-64 µM) directly onto the 13A12 surface
(data not shown). The specific RU for
H-2Kb-CD8f-LZ binding at equilibrium were
determined by subtracting background RU (H-2Kb on the 13A12
surface) from the total RU. The Kd was derived from
a Scatchard plot RU/concentration versus RU and linear regression analysis (Kaleidagraph software). The experiment was repeated three times with CD8-LZ and CD8f-LZ,
and only once with CD8-LZ, CD8f-LZ,
CHO-CD8f-LZ, and CHO-CD8f-LZ. For all BIAcore studies, the H-2Kb -chain (residues 1-274)
and the murine-2-microglobulin (residues 1-99) were expressed as
separate inclusion bodies in Escherichia coli and refolded
in the presence of VSV8 peptide as described earlier (30).
Cytotoxicity Assays--
To obtain N15 CTL, splenocytes were
isolated from Rag2
/ mice expressing a transgenic N15
TCR (36) and stimulated to obtain a short term cytotoxic T cell line as
described previously (37). For stimulation, the splenocytes were
co-cultured in 24-well plates (5 × 106 cells/well)
with irradiated N1 cells (5 × 105 cells/well). N1
cells constitutively present the cognate VSV8 peptide as an expressed
minigene. After 5-7 days of culture with rat-derived concanavalin A
supernatants, N15TCR+ CTLs were harvested and used as
effector cells in the cytotoxicity assay. 1 × 106
EL-4 target cells were labeled with 80 µCi of sodium
[51Cr]chromate for 1 h at 37 °C. After four
washings with RPMI complete, the Kb-expressing EL-4 cells
were resuspended at 50,000 cells/ml in complete RPMI and transferred
into 96-well U bottom plates (100 µl/well). To determine a suboptimal
dose of peptide for inhibitory experiments, peptides were added at
concentrations between 106 and 1012
M in a final volume of 100 µl of complete RPMI and
incubated for 1 h. Finally, CTLs (100 µl) were added at an E:T
ratio of 10:1 or 5:1, and 51Cr release was determined using
standard conditions (36). In the actual blocking experiment, EL-4 cells
were pulsed with VSV8 at 2 × 1010 M. As
a negative control either SEV9 (irrelevant peptide) or only PBS was
added to the wells. Anti-CD8 53.6.72 mAb was used as positive
control, whereas a two-domain recombinant CD4 (rCD4) (38) was used as a
negative control for blocking experiments. 1 µM 53.6.72 mAb was added to CTL cells for 10 min at room temperature. CD8-LZ, CD8-LZ, CD8f-LZ,
CD8f-LZ, CHO-CD8f-LZ, and
CHO-CD8f-LZ were concentrated using a Centricon-10
(Amicon), and 27.5-220 µM (final concentration) of CD8
protein or 27.5-220 µM of rCD4 were added to the antigen
presenting cells 10 min prior to addition of 5 × 104
N15 CTL (in 100 µl)/well. To determine background release, 100 µl
of RPMI completed medium was added to 51Cr-labeled targets.
The maximal 51Cr release was quantitated by adding 100 µl
of 1% Triton X to 51Cr-labeled target cells. After 4 h at 37 °C, the plates were centrifuged, and 50% of the well
solution was removed and counted in a -counter.
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RESULTS |
Design of Secreted Dimeric CD8 and CD8 Ig-like
Ectodomains Using a Leucine Zipper Sequence and Expression in
Lec3.2.8.1 Cells--
A strategy to promote secretion of various
soluble recombinant CD8 co-receptor protein fragments with homogeneous
glycan adducts in the Lec3.2.8.1 eukaryotic cells was developed. To
this end, a set of CD8 co-receptor constructs was produced as depicted
schematically in Fig. 1. For example, the
N-terminal extracellular segment of the mCD8 chain (residues 1-122)
corresponding to the predicted Ig-like domain was fused via a flexible
linker (residues 123-137) and a thrombin cleavage site to either an
Acid-p1 or Base-p1 30-aa-long leucine zipper fragment to form
CD8-Acid (CD8-A) and CD8-Base (CD8-B), respectively (Fig.
1A). Subsequently, cDNAs encoding CD8-A and CD8-B
were transfected into Lec3.2.8.1 cells, and clones secreting the
CD8 ectodomain were screened by ELISA. mCD8-LZ protein was
then affinity purified from the producer clone CD8-22-1 using the
anti-leucine zipper mAb 2H11. As shown in Fig.
2A, under either nonreducing
conditions (NR, lane 1) or reducing conditions
(R, lane 2), the recombinant mCD8-LZ
protein runs in the Coomassie-stained SDS-PAGE gel as two bands
corresponding to CD8-A and CD8-B monomer components with apparent
molecular masses of 29 and 25 kDa, respectively. The difference in
mobility of the CD8 chains is a consequence of the divergent charges
within the appended acid or basic leucine zipper sequences. By this
analysis, the affinity purified mCD8-LZ material is ~90% pure
with a 1:1 ratio of CD8-A and CD8-B. Although not shown, gel
filtration chromatography on Sephadex 75 demonstrated that
mCD8-LZ was dimeric and readily removed the trace amount of
higher molecular mass contaminants and/or aggregates. Moreover, the
expected N-terminal sequence of mCD8-LZ was verified by amino
acid sequencing (not shown).
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Fig. 1.
Constructs of soluble recombinant murine
CD8-LZ,
CD8-LZ,
CD8f-LZ, and
CD8f-LZ.
A, schematic representation of mCD8-LZ showing the
Ig-like domain of CD8 joined to either an Acid-p1 or a Base-p1
peptide via a flexible linker containing a thrombin cleavage site
(indicated by filled triangle) to form a dimer of CD8-A
and CD8-B, respectively. B, CD8-LZ consists of the
Ig-like domain of CD8 joined to Acid-p1 and the CD8 joined to
Base-p1 forming a dimer of CD8-A and CD8-B. C,
CD8f-LZ, the full-length extracellular domain of
CD8 joined to either an Acid-p1 or a Base-p1 peptide via a flexible
linker containing a thrombin cleavage sequence to form a dimer of
CD8f-A and CD8f-B, respectively.
D, CD8f-LZ, the full-length extracellular
domain of CD8 joined to Acid-p1 to form CD8f-A in
noncovalent association with CD8f-B. The positions of
cysteines that were mutated to serines to exclude disulfide bond
formation are marked with crossed dotted lines. The
potential N-linked glycosylation sites (at CD8 aa 43, 70 and 123 and at CD8 aa 13) and O-linked glycosylation
sites (at CD8 aa 124-127, 133, 135, 140, 142, and 143 and at CD8
aa 120, 121, 124, 127, and 128) are schematically represented by
open and filled lollipops, respectively. The
unpaired cysteine (aa 37) in CD8 is denoted (SH).
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Fig. 2.
SDS-PAGE analysis of recombinant CD8
proteins. A, Coomassie staining of affinity purified
CD8-LZ protein run on 12.5% SDS-PAGE gel under nonreducing
(NR, lane 1) and reducing conditions
(R, lane 2). The positions of CD8-A and
CD8-B monomers are indicated. B, Coomassie staining of
affinity purified mCD8-LZ protein run on 12.5% SDS-PAGE gel
under nonreducing (NR, lane 3) and reducing
(R, lane 4) conditions. The positions of CD8-A
and CD8-B monomers were determined by N-terminal amino acid
sequencing as indicated.
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The same strategy was then applied to the expression of the CD8
Ig-like domains in Lec 3.2.8.1. cells. The N-terminal extracellular segment of the mCD8 chain (residues 1-115) corresponding to the predicted Ig-like domain was fused via a flexible linker (residues 116-130) containing a thrombin cleavage site to the Acid-p1 peptide to
yield CD8-Acid (CD8-A) (Fig. 1B). The cDNA
encoding the CD8-A subunit was engineered into the pEE14 vector.
Subsequently, the pEE14 CD8-B and pEE14 CD8-A plasmids were
co-expressed in Lec3.2.8.1 cells, CD8 producing clones were
identified by ELISA and mCD8-LZ protein immunoaffinity purified
using the anti-leucine zipper mAb 2H11. As shown in Fig. 2B,
under nonreducing conditions (NR, lane 3) and
reducing conditions (R, lane 4), the recombinant
mCD8-LZ runs as closely spaced CD8-A and CD8-B monomers at
molecular masses 26 and 25 kDa, respectively. Because the - and -
bands in the CD8-LZ SDS-PAGE could not be separated, CD8-LZ
was first digested with endoglycosidase-H (0.02 unit/mg CD8-LZ
for 2 h at 37 °C) and then resolved by SDS-PAGE. Following
deglycosylation, the CD8-B derivative runs at 20 kDa and the
CD8-A derivative runs at 24 kDa. This differential mobility
permitted N-terminal sequencing of the subunits that confirmed the
identity of the individual chains and suggested a 1:1 ratio of CD8-B
and CD8-A (data not shown). As shown in Fig. 2B, the
immunoaffinity purified mCD8-LZ material is ~90% pure. The
2H11 mAb-purified mCD8-LZ protein expresses the native epitopes
recognized by five distinct anti-CD8 mAbs (53.6.72, 19/178,
H59-101.7, YTS105, and YTS169) (34, 35, 39) and four anti-CD8 mAbs
(53.5.8, H35-17, YTS156.7, and KT112) (34, 35, 39, 40), as measured by
BIAcore binding studies (data not shown).
Expression of the Full-length sCD8 and sCD8
Ectodomains--
The stalk region that connects the CD8 and CD8
Ig domain to the cell membrane contains ~44 aa residues in the chain and ~35 aa residues in the chain (depending on species). To
assess the functional contribution of O-linked glycans in
the stalk region of CD8 to MHC class I binding, we expressed the
soluble full-length sCD8f and sCD8f
in wt CHO cells or the glycosylation mutant Lec3.2.8.1. The former
expression system synthesizes proteins with full-length glycan adducts.
In contrast, Lec3.2.8.1 cells produce glycoproteins with all
N-linked carbohydrates truncated to the Man5
form and O-linked carbohydrate truncated to a single GalNAc
(32).
Several reports indicated that the formation of disulfide-linked
homodimers is extremely inefficient when the entire native ectodomain
of either hCD8 or rat CD8 was expressed. The resulting products contained a mixture of disulfide- and nondisulfide-linked CD8
homodimers, monomers, and aggregates (21-24, 26, 41). To avoid this
complexity, the extracellular segment of the mCD8 chain (residues
1-165) N-terminal to the last extracellular cysteine was fused to
either an Acid-p1 or Base-p1 to form CD8f-A and CD8f-B, respectively (Fig. 1C). Furthermore,
the cysteine residue at aa 151 of CD8 was converted to serine by
polymerase chain reaction mutagenesis to obviate disulfide scrambling.
Both cDNAs were engineered into the pEE14 vector system and
expressed in CHO or Lec3.2.8.1 cells. To express the full-length
mCD8f-LZ, the CD8 cysteine at position 137 was
similarly mutated to serine, and the extracellular segment of the
mCD8 chain (residues 1-149) was fused to an Acid-p1 forming
CD8f-A. The pEE14 CD8f-B and pEE14
CD8f-A plasmids were co-transfected to generate stable cell lines, producing mCD8f-LZ as described under
"Experimental Procedures." Lec3.2.8.1 or wt CHO secreted
mCD8f-LZ and mCD8f-LZ were affinity
purified from culture supernatants using the 2H11 mAb.
As shown in Fig. 3, under nonreducing
conditions, the wt CHO-produced recombinant mCD8f-LZ
runs as one broad band of apparent molecular mass of 30-37 kDa
(Fig. 3, lane 1). By contrast, the Lec3.2.8.1-produced
recombinant mCD8f-LZ protein runs as a discrete band
with a molecular mass of 32 kDa (lane 3). The wt CHO-produced recombinant mCD8f-LZ runs as two rather broader bands
representing CD8f-B and CD8f-A subunits
at molecular masses 35 and 30 kDa, respectively (Fig. 3, lane
2), whereas Lec3.2.8.1-produced recombinant mCD8f-LZ runs as discrete bands of molecular masses
32 and 29 kDa (lane 4). The difference in the molecular
masses of these products reflects the different nature of the
glycosylated adducts. The affinity purified mCD8f-LZ
material is quite pure and shows a 1:1 ratio of CD8f-B
and CD8f-A in Coomassie gel (Fig. 3). The affinity
purified mCD8 molecules bear native epitopes as measured by BIAcore
analysis using multiple mAbs reactive with CD8 co-receptors found on T
lymphocytes (see below).
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Fig. 3.
SDS-PAGE analysis of
CD8f-LZ and
CD8f-LZ proteins
produced in CHO and Lec3.2.8.1 cells. Coomassie-stained gel of
affinity purified CHO-produced CD8f-LZ and
CD8f-LZ protein (lanes 1 and 2,
respectively) and corresponding Lec3.2.8.1-produced
CD8f-LZ (lane 3) and
CD8f-LZ (lane 4) after resolution by
12.5% SDS-PAGE under nonreducing conditions. The positions of
CD8f-A, CD8f-B, and CD8f-A
subunits are indicated for the Lec3.2.8.1-produced material.
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BIAcore Affinity Measurements--
The interactions between
soluble CD8 and MHC class I H-2Kb (complexed to VSV8) were
examined using an SPR biosensor, which allows direct measurement of
kinetic interactions between immobilized and solution-phase molecules
(42). The affinity for H-2Kb-VSV8 was measured using all
six different CD8 protein derivatives (CD8-LZ, CD8-LZ,
CD8f-LZ, CD8f-LZ,
CHO-CD8f-LZ, and CHO-CD8f-LZ). The
proper folding of these soluble CD8 proteins was confirmed by BIAcore
analysis using anti-CD8 (YTS169, 19/178, 53.672, H59-101.7, YTS105)
and anti-CD8 mAbs (YTS156.7, H35-17, 53.5.8, KT112) for binding
studies of the CD8 proteins captured on the 13A12 anti-leucine zipper
mAb surface (data not shown). For affinity measurements, 20 µl of the
individual CD8 or CD8 (at 1 µM) proteins were
first captured by 13A12 on the chip. Subsequently, VSV8/Kb
was injected at 1-64 µM concentrations. Fig.
4 shows a typical sensorgram of the
H-2Kb binding to CD8f-LZ. As a control,
H-2Kb at identical concentrations of 1-64 µM
was injected on a 13A12 mAb surface alone. Although not shown, only
residual nonspecific binding was detected. Because the association and
dissociation phases were too fast to analyze, the equilibrium binding
constant was determined using Scatchard analysis. Independent
experiments have been carried out three times for both CD8-LZ and
CD8f-LZ, and only once for CD8-LZ,
CD8f-LZ, CHO-CD8f-LZ, and
CHO-CD8f-LZ. For Scatchard plots, VSV8/Kb
concentrations of 4-64 µM or 8-64 µM have
been used. Table I summarizes the
affinities of the different CD8 proteins for pMHCI. As shown,
CD8f-LZ has an affinity of 35-75 µM
for H-2Kb-VSV8, whereas that of CD8f-LZ
is comparable at 67 µM. Moreover, CD8 constructs lacking
the C-terminal stalk regions (CD8-LZ and CD8-LZ) have
comparable affinity to the full-length CD8f and
CD8f, implying that the Ig domain is necessary and
sufficient for MHC binding. Moreover, CD8-LZ and CD8-LZ
affinities of 57-86 and 30 µM, respectively, are quite
similar.
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Fig. 4.
Affinity measurement of the
CD8 class I MHC interaction. Shown are
the BIAcore sensorgrams of H-2Kb/VSV8 binding to chip-bound
CD8f-LZ captured by mAb 13A12 (A) and
corresponding Scatchard analysis of H-2Kb/VSV8 binding to
CD8f-LZ (B).
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CD8 on the T cell surface is known to be heavily O-linked
glycosylated in the stalk region and to possess a sialylated Ig domain.
Thus, it was of interest to investigate whether the fully glycosylated
CHO-produced full-length CD8 proteins differ in their affinities for
pMHC relative to Lec3.2.8.1-derived proteins. However, CHO-CD8f-LZ showed an affinity of 64 µM, and CHO-CD8f-LZ showed an affinity
of 22 µM. Based on these results in comparison to the Lec
3.2.8.1. derivatives, there appears to be no difference in affinity
(within a factor of <3) for Kb resulting from the distinct
glycosylation pattern of the CD8 protein.
sCD8 and sCD8 Are Able to Block the Cytotoxic Activity
of N15 CTL--
Next we tested the ability of sCD8 molecules to
functionally inhibit the cytolytic activity of class I
MHC-dependent N15 CTL derived from N15 tg
Rag2/ H-2b mice. In these experiments, CTLs
were added at an E:T ratio of 5:1 to 51Cr-labeled EL-4
cells pulsed with 2 × 1010 M VSV8
peptide, and 51Cr release was determined in the presence or
absence of CHO-CD8f-LZ or
CHO-CD8f-LZ proteins. Anti-CD8 53.6.72 (1 µM) was used as positive control, and a two-domain
recombinant CD4 (rCD4) (38) was used as a negative control for the
blocking experiments. Fig. 5A
shows the results of one representive experiment when the CD8 ectodomain dimers were added to EL-4 cells for 10 min prior to the
cytotoxicity assay. Both CHO-CD8f-LZ and
CHO-CD8f-LZ proteins are able to block the cytotoxic
activity of N15 CTL in a dose-dependent fashion, inhibiting
81.1 and 73.7%, respectively, at a 220 µM concentration.
The concentration of soluble CD8 necessary for 50% functional
inhibition (55-110 µM) correlates with the affinities of
the CD8 dimers for class I MHC as measured by BIAcore. At 110 µM soluble CHO-CD8f-LZ inhibited
slightly better than CHO-CD8f-LZ at the same molar
concentration. Note that anti-CD8 antibody 53.6.72 inhibited killing
by about 95%, whereas rCD4 showed a negligible percentage of
inhibition.
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Fig. 5.
Soluble recombinant CD8 and CD8 dimer fragments
functionally block the cytotoxic activity of N15tg CTL to equivalent
degrees. A, indicated concentrations of wt CHO-produced
mCD8f-LZ and mCD8f-LZ were added to
a cytotoxic assay using N15 CTL effectors generated from splenocytes of
N15 TCR transgenic Rag2/ mice and EL4 cells pulsed with
VSV8 peptide (2 × 1010 M) as target
cells at an E:T ratio of 5:1. The ability of anti-CD8 mAb 53.6.72 (positive control) and a two-domain rCD4 (negative control) to inhibit
lysis is indicated by the solid arrow and open
arrow, respectively. B, 220 µM
concentrations of CHO-CD8f-LZ,
CHO-CD8f-LZ, CD8-LZ, and CD8-LZ as well
as two-domain recombinant CD4 were added into the above
cytotoxicity.
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To address whether the Ig-like domains of the CD8 dimer alone were able
to mediate functional CTL inhibition, we compared the effects of
CD8-LZ and CD8-LZ with those of
CHO-CD8f-LZ and CHO-CD8f-LZ. Fig.
5B shows results for all four CD8 ectodomain dimer fragments
at 220 µM. The CD8 Ig-like domain per se, in
absence of the stalk region and complex glycans, is able to block
cytolytic function. Compared with the CHO derivatives, Lec 3.2.8.1 CD8-LZ only inhibited 36.5%, whereas Lec 3.2.8.1 CD8-LZ
inhibited 48.5%. Thus, the level of CTL inhibition with CD8-LZ
and CD8-LZ is somewhat lower than that observed with the
CHO-produced full-length ectodomain CD8f-LZ and
CD8f-LZ protein. Whether the CD8 stalk region in the
CD8f-LZ and CD8f-LZ derivatives
contributes to the functional inhibition by affecting additional
components of the CTL or target cell surface is unknown. It is also
possible that the greater size of the CHO-CD8f-LZ and
CHO-CD8f-LZ protein relative to the Lec3.2.8.1
CD8-LZ and CD8-LZ proteins creates an additional steric
inhibition that facilitates the observed functional blockade.
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DISCUSSION |
Various CD8 co-receptor ectodomain dimers were produced in soluble
form to investigate the basis for differences in the functions of
and dimers and the contribution of their respective
Ig-like domain and stalk region segments. The yield of immunoaffinity purified protein derived from each construct expressed in Lec3.2.8.1 cells was substantial: 12-15 mg/liter for mCD8 (CD8-LZ) or 8-10 mg/liter for mCD8 (CD8-LZ). The levels of expression of sCD8 compare favorably with previous reports of the expression of
hCD8 (~2 mg/liter of affinity purified CD8) and the
E. coli-produced Ig-like domain of hCD8 (~0.5
mg/liter). The well paired CD8 homodimer expressed in the
glycosylation-defective mutant, Lec3.2.8.1, cell system has already
provided adequate materials for crystallization of the complex of
CD8/Kb (20). The high level expression of mCD8
should allow a detailed structural analysis of CD8/Kb
alone or in complex with a class I MHC-restricted TCR as well.
When the CD8 dimer containing both cysteine residues in the stalk
region was expressed in early studies (22-24, 26, 27, 41), the
majority of the recombinant protein was monomeric, caused by intrachain
pairing of the two cysteine residues in the stalk region (21). In
contrast, the results of SDS-PAGE (Figs. 2 and 3) and gel filtration
here indicate that the subunit products of CD8f-LZ
and CD8f-LZ are in a molar ratio of 1:1 with minor or
no aggregates. This result suggests that the mutation to serine of the
cysteine residue at aa 151 of CD8 or aa 137 of CD8 in the stalk
region avoided the disulfide bond scrambling completely. The yield of
immunoaffinity purified full-length ectodomain using the anti-leucine
zipper mAb, 2H11, was 8-15 mg/liter for CD8f-LZ and
CD8f-LZ expressed in CHO or Lec3.2.8.1 cells.
Moreover, the affinity purified mCD8 molecules are in a native
configuration as measured by BIAcore using five anti-CD8 mAbs and
four anti-CD8 antibodies.
The stalk region connecting the Ig-like domain of mCD8 to the membrane
consists of ~44 aa in the chain and ~35 aa in the chain
(Fig. 1A). Each connecting peptide must be extended because it is rich in proline residues and contains a number of
O-linked glycans. The biological function of these stalks is
unclear as is the precise orientation of the TCR chains relative to the
co-receptor subunits. However, the sialic acid content of CD8
O-linked glycans decreases significantly on thymocytes and
activated T cells compared with the levels found on resting T cells
(43-45), a phenomenon not observed for the CD8 chain. The results
imply that the ability of CD8 to vary its overall charge and glycan
size may have important consequences for CD8-TCR interaction. To
examine the functional contribution of the O-linked glycan
in the stalk region of CD8, we expressed the soluble full-length
sCD8 and sCD8 in wt CHO cells or in the glycosylation
mutant Lec3.2.8.1, allowing a direct comparison of the importance of
the complex sugars to the MHC binding function of CD8. A cytotoxicity
assay utilizing the N15 CTL derived from N15 tg Rag2/
H-2b mice then assessed the ability of soluble CD8
molecules to inhibit the cytolytic activity of class I
MHC-dependent N15 CTL. The results of two independent
experiments show that CHO-produced CD8f-LZ and
CHO-CD8f-LZ proteins are able to block the cytotoxic
activity of N15 CTL in a dose-dependent fashion, with no
significant difference between the CHO-CD8f-LZ and
CHO-CD8f-LZ proteins. This result implies that the
binding of soluble CD8 or CD8 to MHC class I molecules on
target cells must be equivalent. These results are consistent with the
BIAcore analysis showing similar Kb binding for CD8
and CD8 dimers (Table I). Moreover, both the Lec3.2.8.1-produced
CD8 and CD8 Ig-like domains, without the stalk region, are
able to block cytolytic function.
The affinity of CD8 to H-2Kb-VSV8 has been measured using
all six different CD8 constructs produced (Table I).
CD8f-LZ has an affinity of ~62 µM
(n = 4) for H-2Kb-VSV8, and
CD8f-LZ show a slightly higher affinity of ~44
µM (n = 2). CD8 constructs lacking the
C-terminal stalk regions, CD8-LZ and CD8-LZ, do not appear
to have lower affinity (~67 µM (n = 3)
and 30 µM (n = 1)) than their full-length
protein counterparts (Table I). These affinities are comparable with
those determined previously by Garcia et al. (46). In that
report, the full-length ectodomains of mCD8 and mCD8
expressed with a histidine tag in a Drosophila system reveal
a moderate affinity (~39 and ~11 µM, respectively) to
MHC class I molecules loaded with different antigenic peptides, with
the CD8 heterodimer having a slightly better affinity. However,
the results are substantially different from the affinity measured by
Wyer et al. (28). Utilizing the E. coli-expressed
recombinant Ig-like domain of hCD8, the affinity of hCD8 to
pMHCI was determined to be greater than 200 µM (28). Species differences, allelic differences, the absence of the mucin-like stalk region for sCD8, and/or the presence of aggregated materials might explain these discrepancies. In the present expression system, Lec3.2.8.1-produced CD8 protein shows very little difference from the
CHO-produced CD8. These findings imply no substantial difference in
affinity because of the glycosylation pattern of the stalk region of
the CD8 or CD8. Furthermore, they show that greater
Dimers*
,
TOP
ABSTRACT
INTRODUCTION
