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, But Not Mouse CD8, Can Be Expressed in the Absence of CD8
as a Homodimer1
*
Department of Laboratory Medicine and Section of Immunobiology, and
Department of Genetics, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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polypeptide chains and
heterodimers of CD8- and CD8
-chains. The function of the
CD8-chain for binding to MHC class I and associating with the
tyrosine kinase p56lck was demonstrated with
CD8 homodimers. CD8 functions as a better coreceptor, but
the actual function of CD8 is less clear. Addressing this issue has
been hampered by the apparent inability of CD8 to be expressed
without CD8. This study demonstrates that human, but not mouse,
CD8 can be expressed on the cell surface without CD8 in both
transfected COS-7 cells and murine lymphocytes. By creating chimeric
proteins, we show that the murine Ig domain of CD8 is responsible
for the lack of expression of murine CD8 dimers. In contrast to
CD8, CD8 is unable to bind MHC class I in a cell-cell
adhesion assay. Detection of this form of CD8 should facilitate studies
on the function of the CD8 -chain and indicates that caution should
be used when interpreting studies on CD8 function using chimeric
protein with the murine CD8 Ig domain. In addition, we
demonstrate that the Ig domains of CD8 are also involved in
controlling the ability of CD8 to be expressed. Mutation of B- and
F-strand cysteine residues in CD8 reduced the ability of the protein
to fold properly and, therefore, to be expressed. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
heterodimers of CD8 and CD8. The CD8 homodimer is
exclusively expressed on subsets of intraepithelial lymphocytes of the
intestine (1) and on human NK cells (2).
The crystal structure of CD8 demonstrates that the structure of
the N-terminal 114 aa of each subunit is typical of a light-chain
variable domain and that the two Ig domains of the dimer forms a
structure very similar to an Ab-variable region (3). The
connecting peptide region is a 48-residue linker that precedes the
transmembrane sequence and has an extended structure (4).
This region contains two cysteine residues that are thought to form
disulfide bridges critical for the formation of CD8 dimers and also
contains a number of O-linked glycosylation sites. All Ig
molecules also contain a canonical disulfide bond between cysteine
residues on the B- and F-strands. CD8 has both of these conserved
cysteines and also contains an additional conserved cysteine residue on
the C-strand. Biochemical and crystal structure analysis provided
contradictory results regarding which two cysteines were involved in
the formation of the disulfide bridge. All three crystal structures
involving CD8 (mouse and human) showed that the B- and F-strand
cysteines were the ones making the disulfide bridge (3, 5, 6), whereas two separate biochemical studies have demonstrated
that the B- and C-strand cysteines made the disulfide bond in CD8
(7, 8). In this study, we demonstrate that the B- and
F-strand cysteines are critical for the proper folding and
expression of dimeric CD8 using a mutational analysis.
Functional differences exist between homodimer and heterodimer forms of
CD8, although the mechanisms responsible for these differences are not
fully understood. It has been shown that both molecules are critical
for T cell development (9, 10, 11) and several studies
indicate that CD8 functions as a better coreceptor with the TCR
than CD8. One hypothesis to explain the improved coreceptor
activity of CD8 was that CD8 had a stronger affinity for
MHC class I. However, we showed that, in the absence of the TCR,
CD8 does not bind better to MHC class I than CD8
(12). In addition, we performed a mutational analysis to
determine the orientation of CD8 relative to MHC class I. The
crystal structures of CD8 and MHC class I demonstrated that the
contribution of the two CD8 domains was asymmetric, with one domain
contributing more of the contact residues than the other
(5). We found that, for human CD8, CD8
corresponded to the domain making 70% of contact and CD8
corresponded to the one making 30% of the contact. Thus, this
supported the hypothesis that CD8 played a lesser role in the
interaction of CD8 with MHC class I (13).
Studies on the function of CD8 have been hindered by the apparent
inability of CD8 to be expressed without CD8
(14, 15, 16). In this study, we demonstrate that human, but
not mouse, CD8 can be expressed on the cell surface as a homodimer.
Creation of chimeric molecules to map the region of the CD8 molecule
responsible for this difference demonstrated that only molecules with
human CD8 Ig domain could be expressed on the cell surface. This is
consistent with studies on TCR V-chains in which the Ig domains have
also been shown to be critical for those V-chains that are able to
form homodimers (17, 18). The expression of CD8
homodimers has allowed us to directly examine the ability of CD8
homodimers to bind to MHC class I. Results presented here illustrate
that, in the absence of CD8, CD8 cannot mediate binding to MHC
class I.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All cell lines were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U penicillin/ml, 100 µg streptomycin, and 10% FBS. The cell lines used included the EBV-transformed B cell line UC transfected with luciferase gene (19) and the monkey kidney cell line COS-7 (20).
Construction of chimeric mouse/human CD8 constructs
To determine which domain of human CD8
(hCD8)3 was
responsible for its ability to be expressed in the absence of CD8,
two CD8 chimeric constructs were created. One contained the Ig
domain of hCD8 (aa 1–116) and the stalk, transmembrane, and
cytoplasmic domain of mouse CD8 (mCD8) (aa 118–193; chimera A).
The other chimeric construct contained the mCD8 Ig domain (aa
1–117) and the remainder from hCD8 (aa 117–201; chimera B). The
constructs were generated using overlapping PCR. The first round of PCR
involved a standard outside primer (T3 or KS) and an internal primer
containing a sequence matching the end of the Ig domain of the template
and the beginning of the stalk of the other CD8. The internal
primers used for chimera A were 5' sense primer (CAG CTG AGT GTG GTT
GAT TTC CTT CCT ACA ACT GCC CCA AC) and 3' antisense primer (GTT GGG
GCA GTT GTA GGA AGG AAA TCA ACC ACA CTC AGC). For chimera B, they were
5' sense primer (GCT GAC TGT GGT TGA TGT CCT TCC CAC CAC TGC CCA G) and
3' antisense primer (GGC TGG GCA GTG GTG GGA AGG ACA TCA ACC ACA GTC
AG). The second round of PCR linked the appropriate Ig domain and
remaining regions together using the same external primers in a
reaction containing PCR products from the first round as templates.
Both constructs were sequenced and subcloned into the expression vector
pcDL-SR-296 (21).
Transfection of COS-7 cells
COS-7 fibroblasts were transfected using a modification of a
previously described method (22). Briefly, an expression
vector containing human and mouse CD8 cDNA (CD8 wild type (WT) or
mutants (MTs) and/or CD8 WT or chimeras) were mixed with 8 µl of
lipofectamine per 100 µl of the serum-free medium, Optimem (both from
Life Technologies, Grand Island, NY) for 30 min at room temperature.
This lipofectamine mixture was added to a 35-mm dish of nearly
confluent COS-7 cells. Transfection was stopped after 5 or 18 h by
replacing the lipofection mix with 2 ml of fresh medium containing 10%
FCS. Cells were fed again after 24 h, and after an additional
24 h the dishes were either analyzed for cell-surface expression
of CD8 or used in the adhesion assay.
FACS analysis of cell-surface molecules
For the cell-surface expression of hCD8, COS-7 transfectants
were stained with primary mAbs OKT8 (Coulter, Westbrook, ME), which
recognizes an epitope on CD8; 5F2, which recognizes an epitope on
CD8; and 2ST85H7 (Immunotech, Westbrook, ME), which recognizes a
conformational epitope formed by CD8. Expression of mCD8 was
examined with PE-conjugated forms of clone 53.6.7 (PharMingen, San
Diego, CA) to detect mCD8 and with PE-conjugated forms of clone
CT-CD8b (Caltag, Burlingame, CA) to detect mCD8.
Cells from the blood of hCD8 transgenic mice were stained with the
PE-conjugated clones described above for detecting mCD8 and mCD8.
In addition, a FITC-conjugated form of RM 4-5 (PharMingen) was used to
detect mCD4, and a biotinylated form of 5F2 (produced on request by
Immunotech, Marseille, France) with streptavidin-PE (PharMingen) as the
second step was used to detect hCD8.
Adhesion assay of class I+ cells to CD8-transfected COS-7
The assay used was a modification of the method previously
described in detail (22). COS-7 cells expressing CD8 were
tested for their ability to bind an MHC class I+
B cell line, UC (19). The HLA Ags expressed by the UC
cells were A1, A2, B5, B57, Cw4, DR7, DQ2, and DQ3. These cells
constitutively expressed the firefly luciferase gene under the control
of the CD8 promoter (23), a property which was utilized
to measure binding. Transfected COS-7 cells were washed once with PBS,
and 107 UC cells were added to each 35-mm dish.
The cells were incubated for 1 h at 37°C and the UC cells were
aspirated off. After several washes with PBS, the number of bound cells
was determined by measuring the amount of luciferase activity in the
cell extract.
Metabolic radiolabeling and immunoprecipitation
COS-7 cells transfected with vector alone, CD8, or both
CD8 and CD8 were metabolically labeled with
35S-labeled cysteine and methionine 24–48 h
posttransfection, and then they were chased and extracted for
immunoprecipitation. Cells were deprived of cysteine and methionine for
1 h in methionine- and cysteine-free RPMI 1640 (ICN, Irvine, CA)
containing 3% dialyzed FCS, and then they were pulsed with 0.5 mCi
35S-labeled cysteine and methionine
(TRAN-35S-LABEL; ICN) for 1 h at 37°C. The
labeled cells were washed with PBS and then lysed in 150 mM NaCl, 10 mM
Tris (pH 7.4) containing 1% Triton X-100, 0.5 mM PMSF, 0.2 trypsin
inhibitor units/ml aprotinin, 0.1 mM tosyl lysyl chloromethyl ketone, 1
µg/ml pepstatin A, and 5 mM iodoacetamide (pH 8) for 30 min at 4°C.
Nuclei were removed by centrifugation for 15 min at 13,000 rpm.
Supernatants were then precleared four to six times (30 min, overnight)
with protein G zysorbin (Zymed, San Francisco, CA) and then
precipitated with 5F2 (CD8) for 1 h and with protein
G-Sepharose (Zymed) for an additional hour. Pellets were washed three
times with lysis solution and run on SDS-PAGE under nonreducing and
reducing conditions. Gels were then dried and exposed to Kodak
(Rochester, NY) Biomax MR film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expressed in the absence of CD8
Transfection of an expression vector containing human CD8 cDNA
alone was sufficient for cell-surface expression of CD8 on COS-7
cells as detected by staining with the hCD8-specific Ab 5F2 (Fig. 1
). These cells did not stain positively
with the anti-CD8 Ab OKT8 (Fig. 1) or the anti-CD8 Ab
2ST85H7 (data not shown), indicating the absence of CD8 and
CD8 on the cell surface. The level of expression of CD8 was
similar to that of CD8 heterodimer, although twice as much CD8
DNA was transfected in the case of CD8 alone compared with the
results from using CD8 (each plate was transfected with a total
of 2 µg of DNA). Unlike hCD8, expression of mCD8 was only
detected when cotransfected with mCD8, indicating apparent species
differences in the expression of CD8.
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to be expressed in vivo in the absence of CD8
was also seen in transgenic mice with a 95-kb human genomic fragment
containing the entire hCD8 gene. Using a biotinylated form of 5F2,
we were able to detect hCD8+,
mCD8-, mCD4+ cells in
peripheral blood (Fig. 2), lymph node,
and thymus (data not shown). These data indicate that the hCD8 gene
can be expressed in lymphoid cells in the absence of CD8. Expression
of the endogenous mCD8 was not detected in these CD4-positive
cells.
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To determine whether hCD8 could bind MHC class I-positive cells
(UC-luciferase) in the absence of CD8, we performed a binding assay.
Fig. 3 demonstrates that no significant
binding of UC-luciferase cells was detected to COS-7 cells expressing
only hCD8. In contrast, binding was detected to COS-7 cells
expressing CD8. Expression of hCD8 and hCD8 was as seen
in Fig. 1
.
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expressed on COS-7 cells
Because CD8 is normally expressed as a dimer, we wanted to
examine the form of hCD8 expressed on the COS-7 cells in the absence
of CD8. To do this, we metabolically labeled the cells with
[35S]L-cysteine and
[35S]L-methionine
(TRAN-35S-LABEL) and performed an
immunoprecipitation with an Ab against CD8, 5F2. The proteins were
run on SDS-PAGE under reducing and nonreducing conditions, and the gel
was dried and exposed to x-ray film. Fig. 4 demonstrates that when CD8 is
precipitated from COS-7 cells expressing CD8 or CD8 under
reducing conditions, there are three bands at 
60, 50, and 25 kDa.
The bands at 60 and 50 kDa are likely to be homodimers because when
the samples are run under reducing conditions these bands disappear and
new bands appear (presumably monomers) at 30 and 25 kDa. The 50-kDa
protein is probably a nonglycosylated form of CD8 because the
glycosylated form of CD8 is 64 kDa (24).
Interestingly, CD8 monomers of the nonglycosylated form are also
present in the nonreduced lane. However, this is not unique to CD8
because these monomers can also be seen in the samples precipitated
from CD8 transfectants. Similar results were obtained for
CD8 transfectants when immunoprecipitation was conducted with an
Ab against CD8 (results not shown).
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is responsible for homodimer expression
Chimeric CD8 constructs were generated with human and mouse
CD8 to determine which region of hCD8 was responsible for its
ability to form CD8 homodimers. Using flow cytometry we were able
to demonstrate that chimera A containing the Ig domain of hCD8 and
the stalk, transmembrane, and cytoplasmic domain of mCD8 was
sufficient to allow homodimers to form. Chimera B (containing the
mCD8 Ig domain with the stalk, transmembrane domain, and cytoplasmic
domain of hCD8) was not expressed unless cotransfected with mouse or
human CD8 (Fig. 5). This indicates
that the Ig domain of CD8 is the critical factor in determining
whether the protein can be expressed as a homodimer in the absence of
CD8.
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To determine which of the three conserved cysteines in the Ig
domain of CD8 form the disulfide bridge critical for the proper
folding of the protein, we conducted site-directed mutagenesis of all
three cysteines (Cys22 on the B-strand,
Cys33 on the C-strand, and
Cys94 on the F-strand). Fig. 6A shows that only the B- and
F-strand MTs had an effect on the ability of CD8 to be expressed
efficiently. These mutations dramatically reduced the level of
expression of CD8 on the cell surface. Results shown are for
staining with OKT8; however, a panel of hCD8 Abs were examined
(G10.1, Leu-2a, and 66.2) and all were found to stain in a manner
similar to that shown. However, in the presence of hCD8, the
percentage of cells expressing MT CD8 increased by 2-fold (Fig. 6B). This effect was observed with both the B- and F-strand
CD8 MTs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, hCD8 can be expressed on the cell surface of
both COS-7 cells and murine lymphocytes in the absence of CD8. This
is consistent with previous studies with mCD8, which show that cell
lines transfected with mCD8 contain mRNA but not cell-surface
expression in the absence of CD8 (14, 16). However, our
results are discordant with previous studies reporting that hCD8
also required CD8 to be expressed. A probable explanation for this
apparent contradiction is that in previous studies the Ab 2ST85H7 was
used to detect expression of hCD8 (15, 24). It is now
known that this Ab will only detect expression of the CD8
heterodimer, whereas 5F2, the Ab we used that was developed later,
detects CD8 alone (25). It is unlikely that failure to
detect mCD8 in the absence of mCD8 is due to the Abs only
recognizing the heterodimer because the anti-mCD8 Ab 53.3.8 was
shown to recognize an epitope retained on the mCD8 subunit after
dissociation of the heterodimeric mCD8 complex
(14).
In addition to expression of CD8 on COS-7 cells, in vivo evidence of
expression of hCD8 in the absence of CD8 comes from mice
expressing a hCD8 transgene. Despite otherwise tissue-specific
expression of the transgene, we could detect hCD8 expression on a
subpopulation of CD4+ lymphocytes, albeit at a
lower level than that found on mCD8 cells. Previous experiments to
ascertain whether mCD4 cells expressed the hCD8 transgene failed to
detect this low-level expression. The reason for the discrepancy
appears to be the staining reagents. Previously, we used a
streptavidin-Red 670 reagent that stained the cells with a lower
intensity than the streptavidin-PE used in this experiment
(26).
Because the present transgenic mouse data indicate that hCD8 can be
expressed on murine lymphoid cells in the absence of CD8, what
remains to be determined is whether such expression exists on human
lymphoid cells. Staining of human blood did not detect any cells that
were CD8-CD8+
(results not shown). However, this does not rule out the possibility
that CD8 homodimers are expressed on the same cells as CD8
homodimers and CD8 heterodimers. It will be important to
determine whether this form exists on thymocytes or on mature T cells,
either resting or activated.
Using CD8 chimeras, we found that the Ig domain was critical for the
ability of hCD8, to form homodimers because only chimeras with
hCD8 Ig domain were expressed without CD8. Comparison of the Ig
domains of human and mouse CD8 indicate some potential differences
that could affect the ability of these domains to pair properly. The
C'- and G-strand bulges and adjacent loops, regions that are important
in dimerization, contain charge differences between the two species.
CD8 contains the conserved sequence in the G-strand bulge (F-G-X-G),
but in the mouse form the X is threonine and in the human form X is
lysine. In addition, comparison of the C-C' loop between the two
species also shows that there are a number of charge differences with
the mCD8 containing four charged residues (both positive and
negative) that are conserved in the rat but not in the human.
Mutational analysis is required to determine whether these charge
differences are responsible for the disparity reported here between
mouse and human CD8.
The finding that murine Ig domains do not appear to be able to
associate properly calls into question the physiological relevance of
using chimeric molecules to study the function of mCD8. Chimeric
proteins have been created that contain mCD8 transmembrane and
cytoplasmic domains with the extracellular domain of mCD8
(27). These chimeras are reported to be expressed at low
levels on T cell hybridomas, and presumably they homodimerize due to
the transmembrane domain of CD8 that has been shown to promote dimer
formation (28). Results from the study by Wheeler et al.
(27) suggest that chimeric CD8 can mediate interaction
with MHC class I independent of CD8. Our results with the hCD8
homodimers do not support this observation. Despite the relatively high
levels of hCD8 expressed on COS-7 cells, we were unable to detect
binding to MHC class I-positive cells. It is likely that the CD8
homodimers in the mouse chimera used by Wheeler et al. do not associate
properly because the Ig domains of mCD8 do not normally form dimers.
This could explain their finding that chimeric CD8 homodimers
bound better to a form of MHC class I containing a mutation in the 3
domain of MHC class I (27) that was previously shown to be
a critical residue for CD8-MHC class I interactions in both binding and
CTL assays (29).
Consistent with the importance of the Ig domains of CD8 for dimer
formation is our finding that mutant cysteine residues in the B- and
F-strands of the CD8 Ig domains resulted in a decrease in CD8
expression, whereas mutation of both cysteines in the stalk had no
significant effect (our unpublished observations). Absence of both
cysteines in the stalk of CD8 did result in a small decrease in the
expression of CD8 heterodimers (our unpublished observations),
indicating that these residues may play more of a role in the formation
of heterodimers. The B- and F-strand mutations most likely affected the
ability of the protein to properly fold, hindering their ability to
form dimers and hence their ability to be expressed on the cell
surface. However, we were able to rescue some expression of MT CD8
as a heterodimer with CD8 WT because CD8 was properly folded and
could bring more CD8 to the cell surface.
An interesting point to consider in light of the data we have presented
here on the expression and function of CD8 is how the CD8 molecule
may have evolved. The two genes for CD8 and are 36 (in mice) or
56 (in humans) kb apart and probably originated from a common ancestor.
Because CD8 can bind to MHC class I in the absence of CD8 and is
able to associate with the tyrosine kinase
p56lck, one could assume that this molecule was
the first gene. Further support for this hypothesis comes from the
expression patterns of the two proteins. CD8 homodimers, but not
CD8 heterodimers, are expressed on human and rat NK cells as well
as on 
T cells, all of which are involved in the innate immune
response. Once the gene duplicated, CD8 may have evolved for
specific immune responses to enhance the ability of CD8 to act as a
coreceptor with the TCR because CD8 does function as a better
coreceptor (30, 31). Why mCD8 has evolved so that it is
not expressed in the absence of CD8, whereas hCD8 has not,
remains to be determined.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paula B. Kavathas, Department of Laboratory Medicine, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208035, New Haven, CT 06520-8035. E-mail address:
3 Abbreviations used in this paper: hCD8, human CD8; mCD8, mouse CD8; WT, wild type; MT, mutant.
Received for publication August 2, 1999. Accepted for publication October 29, 1999.
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References |
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