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*
Department of Medicine, Division of Immunology and Rheumatology, Stanford University Medical Center, Stanford, CA 94305; and
Division of Basic Immunology, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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molecules (extracellular
CD8ß, transmembrane and cytoplasmic CD8) also results in
enhancement of T hybridoma responses to alloantigen, suggesting that at
least part of CD8ß’s ability to influence responses similar to those
of mature CD8+ T cells is mediated by its
extracellular domain. Current data suggest that CD8ß-mediated
response enhancement proceeds through mechanisms similar to those
mediated by CD8, i.e., interacting with MHC class I and stabilizing
CD8-associated Lck activity. In this study we present evidence that the
extracellular portion of CD8ß is capable of independent interaction
with MHC class I/ß2m dimers in the absence of CD8. In
addition, CD8ß may enhance interaction with MHC class
I/ß2m when associated with CD8. We also present
evidence from T hybridoma responses suggesting that the extracellular
portion of CD8ß is uniquely capable of efficient interaction with the
TCR/CD3 complex and may couple the TCR/CD3 complex to other surface
components capable of enhancing TCR-mediated signals. This represents
the first evidence that a critical coreceptor function can be
preferentially associated with the CD8ß subunit. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains or as a
disulfide-linked heterodimer of - and ß-chains on mature MHC class
I-restricted T cells, thymocytes, and transfectants (1). CD8ß is
normally only detected when it is coexpressed with and disulfide-linked
to CD8, whereas CD8 can be readily detected without CD8ß.
Although the vast majority of peripheral MHC class I-restricted T cells
express CD8ß heterodimers (2, 3), most work has focused on the
function of CD8 in its homodimeric form, which is expressed
predominantly on some 
T cells as well as on transfected cells
and human NK cells (4, 5). Homodimers of CD8 are known to bind to
nonpolymorphic residues in the 3 domain of MHC class I molecules
(6, 7, 8, 9). CD8 is also responsible for direct association via its
cytoplasmic tail with the protein tyrosine kinase Lck, which is
critical for T cell activation (10, 11, 12, 13). T cell lines expressing
CD8ß molecules are capable of enhanced interaction with MHC class
I on APCs upon activation through TCR/CD3 (14). CD8 can increase
adhesion between the T cell and its target by potentially binding to
all MHC class I on target cells, but enhances T cell responses
optimally when bound to the same MHC/peptide complex as the TCR (15, 16). Thus, by acting as a coreceptor with the TCR for MHC class I, CD8
spatially juxtaposes Lck to components that may serve as its
substrates, thereby potentiating a signaling cascade. Recent studies
suggest that coligation of class I MHC by CD8 can increase the affinity
of the TCR for Ag/MHC, and this effect is diminished by addition of
anti-CD8 or anti-CD8ß mAb (17). Coreceptor activity of CD8
is, therefore, dictated minimally by interaction with both class I
MHC/ß2m and Lck. Optimal coreceptor activity may
also depend upon efficient molecular interaction of CD8 and TCR/CD3
molecular complexes, and it is possible that CD8 and CD8ß may
influence such interactions to different degrees.
Relatively little is known about the contribution of CD8ß to CD8
coreceptor function. Positive and negative thymic selection are
impaired within the CD8+ SP lineage in mice deficient in
CD8ß protein expression, suggesting that CD8ß can be involved in a
presumptive coreceptor activity (18, 19, 20). CD8ß expression results in
enhanced IL-2 production and/or altered specificity in allogeneic class
I MHC-restricted T cell hybridomas (21, 22), so it is reasonable to
assume that CD8ß could influence coreceptor function during T cell
development and activation in the periphery. In addition, transgenic
mice expressing a cytoplasmic tailless form of CD8ß are deficient in
CD8+ SP-lineage T cells (23), suggesting that some
developmental effects of CD8ß (i.e., those affecting positive
selection of CD8+ SP thymocytes) may require the CD8ß
cytoplasmic tail or factors associated with this domain. The
cytoplasmic tail of CD8ß may also stabilize the interaction between
CD8 and Lck and contribute to increased CD8-associated kinase
activity (24). It is plausible that the cytoplasmic tail of CD8ß and
possibly Lck are important for CD8ß’s role in coreceptor activity
during development and upon activation of the T cell. However,
expression of chimeric CD8ß- molecules (extracellular CD8ß,
transmembrane and cytoplasmic CD8) also results in enhancement of T
hybridoma responses to alloantigen (21), suggesting that at least part
of CD8ß’s ability to influence responses similar to those of mature
CD8+ T cells is mediated by its extracellular domain.
Extracellular CD8ß-mediated enhancement of specific responses in
hybridomas has been proposed to involve at least two nonmutually
exclusive mechanisms. Extracellular CD8ß could increase adhesion
between T hybridoma and stimulator cells via direct interaction with
nonpolymorphic residues on the MHC class I alloantigen in much the same
manner as CD8 interacts with MHC class I (21, 22). Adhesive
interaction between CD8ß and MHC class I proteins might additionally
account for the activated binding observed on CD8ß+ T
cell lines (14). Alternatively or in addition, CD8ß might lower a
response threshold for T cell activation, perhaps by physically
interacting with the TCR/CD3 complex and/or efficiently coupling
TCR/CD3 to factors that are capable of enhancing specific signaling
(21). CD8ß is known to be physically altered by differential
O-linked glycosylation on immature thymocytes as well as on
activated peripheral T cells (25), whose coreceptor activity is
probably influenced by CD8ß. Structural alterations such as
differential glycosylation might be expected to alter functional
associations (i.e., with TCR/CD3) mediated by the extracellular domain
of CD8ß. In this study we present evidence that CD8ß is capable of
independent interaction with MHC class I/ß2m dimers. In
addition, we present evidence suggesting that CD8ß uniquely mediates
efficient interaction with the TCR/CD3 complex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HTB-157.7 hybridoma cells were provided by Dr. J. Schneck (Johns
Hopkins Medical School, Baltimore, MD) with permission from Dr. M. Dorf
(Harvard Medical School, Boston, MA). L-Kb cells
express the wild-type H-2Kb gene and protein and were
provided by Dr. J. Schneck. 28Kb cells were derived by
transfecting Ltk- cells with a hybrid gene encoding the
1 and 2 domains of H-2Kb and the 3 domain of
H-2Dd; 29Kb cells are Ltk- cells
transfected with the hybrid Kb/Dd gene, which
additionally harbors a mutation within the 3 domain at residue 227
(Glu to Lys) (6, 7).
Transfectants expressing SCßDds (a soluble H-2Dd molecule tethered to ß2m) (26) were provided by Dr. D. Margulies (National Institutes of Health, Bethesda, MD). The presence of the secreted protein in supernatants was monitored before use by SDS-PAGE analysis and compared with that in supernatants of untransfected cells.
Surface expression of transfected genes and TCR/CD3
was detected by
cytofluorometry before functional analysis using the following Abs:
53-6.72 (rat IgG2a anti-mouse CD8), YTS-156.7.7 (rat IgG2b
anti-mouse CD8ß), H57-597 (hamster IgG anti-mouse TCRCß),
and yCD3.1/145-2C11 (hamster IgG anti-mouse CD3). In addition,
14.8 (rat IgG2b anti-mouse CD45RA) Ab was used as a negative
control for SCßDds inhibition assays. CD45RA is not
expressed by untransfected or CD8ß- or CD8ß-' transfectants
of HTB-157.7.
All mAb were purified from hybridoma culture supernatants by passage over protein G-Sepharose columns, washing with PBS, and elution with 0.1 M glycine (pH 2.7). Fractions were collected and neutralized with a 1/5 dilution of 2 M Tris-Cl (pH 10), assayed for protein content, and dialyzed three times against PBS.
Vectors and transfections
cDNAs encoding mouse CD8, CD8', and CD8ß were previously
subcloned into the expression vector pHßAPr-1-neo (27). The hybrid
CD8ß-' gene construct (pßRV') was engineered by creating
EcoRV sites at the extracellular/transmembrane junctions of
the mouse CD8' and CD8ß cDNAs and by excising and replacing the 3'
CD8ß EcoRV-SalI (vector) fragment with that
from CD8'. HTB-157.7 transfectants expressing CD8', CD8' plus
CD8ß, or hybrid CD8ß-' cDNAs were generated as follows (see
Tables I and II for expression profiles). CD8, CD8', CD8ß,
CD8ß-, and CD8ß-' DNAs were transfected alone (CD8,
CD8', CD8ß-, and CD8ß-' DNAs, 30–60 µg) or in
combination (CD8 plus CD8ß cDNAs, or CD8' plus CD8ß cDNAs, at
a weight ratio of 1:5 CD8 or CD8':CD8ß DNA, 80–90 µg) into
8 x 106 HTB-157.7 cells by electroporation at 300 V.
Transfected cells were subdivided into individual wells and selected in
medium containing G418 (1.7 mg/ml) with no further cloning. Surface
expression of CD8, CD8ß, and CD3 or TCRß on transfectants was
examined by FACS staining, and transfectants were chosen for analysis
based on FACS results. Transfectants were subdivided immediately before
assay into equivalent portions and plated onto L-Kb,
28Kb, or 29Kb stimulator cell transfectants or
onto plate-bound anti-CD3 mAb (see Fig. 4
).
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Stable HTB-157.7 transfectants (CD8, CD8', CD8,
CD8', CD8ß, CD8ß-, CD8ß-') were analyzed for surface
expression of CD8 (mAb 2.43 or 53-6.72) (28), CD8ß (mAb 53-5.8)
(27), YTS-156.7.7 (H. Waldmann, Oxford University, U.K., unpublished
observation), and CD3 (mAb 145-2C11) (29). Primary Ab was incubated
with cells on ice for 15 to 45 min, followed by washes with PBS and
10% FCS, secondary staining with fluorescein-conjugated mouse
anti-rat IgG, washing, and analysis by flow cytometry. Alloantigen
expression on stimulator L cell transfectants was confirmed initially
with mAbs 20-8-4 (anti-H-2Kb,bm10,bm1) (30).
Stimulator cells were irradiated with 4500 rad, plated into microtiter
wells in 100 ml of RPMI medium at the indicated densities, and allowed
to settle for 3 to 18 h at 37°C. One hundred microliters of
HTB-157.7 cells (untransfected or transfected) at 106
cells/ml were added to triplicate wells and incubated at 37°C for 18
to 24 h, at which time 100 µl of supernatant was removed for
IL-2 quantitation using the IL-2-dependent line HT-2 (31).
Transfectants were stimulated on the densities of stimulator cells
indicated in the figures. Equal aliquots of transfectants were analyzed
simultaneously on 29Kb (denoted by "m" after clone
designation) or 28Kb stimulators, and, where indicated, on
plate-bound anti-CD3 mAb or L-Kb. All cells were
analyzed a minimum of four times with similar results, with a
representative assay shown. Purified protein or supernatants used for
inhibition studies were added to wells before introduction of HTB-157.7
cells at the indicated concentrations. For inhibition studies, the
following reagents were additionally added to triplicate wells for each
density of L-Kb: 4.0 µg/well (10 µl) of an irrelevant
mAb, 14.8 (anti-CD45RA, not expressed by untransfected or
CD8ß- transfectants of HTB-157.7; data not shown), or 30 µl of
supernatant from L cell transfectants that secrete SCßDds
(SCDd). Supernatants were removed after incubation at 37°C in 5%
CO2 overnight and were assayed for IL-2 release measured by
[3H]thymidine incorporation by HT-2 cells (31).
Transfectants were divided and analyzed on L-Kb and
plate-bound yCD3.1 mAb at the concentrations indicated
(L-Kb densities were 1.9, 5.6, 16.7, and 50 cells/well
x 103 where not otherwise indicated) in Figure 1. Similar
results were obtained with plate-bound 145-2C11 and H57-597 in addition
to yCD3.1 (shown) mAbs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To directly examine the possibility that the extracellular domain
of CD8ß interacts with MHC class I in a functionally relevant manner,
we examined the ability of a soluble nonantigenic class I heavy chain
(H-2Dd) covalently tethered to the ß2m
light chain (SCßDds) (26) to inhibit specific responses
by CD8ß- and CD8ß-' transfectants of the T cell hybridoma
HTB-157.7 (32). HTB-157.7 is a CD8ß-,
CD4-, MHC class I-restricted allogeneic T cell hybridoma
that specifically recognizes H-2Kb or derivative molecules
on the surface of transfected L cell stimulators (L-Kb) and
responds to these stimuli by producing IL-2 (32, 33). We previously
demonstrated that CD8ß transfectants of HTB-157.7 produce more
IL-2 in response to limiting amounts of L-Kb than do CD8
transfectants expressing closely matched levels of CD8 and CD3
(21). We generated HTB-157.7 transfectants expressing CD8,
CD8ß, or chimeric CD8ß- as previously described (21) as well
as transfectants expressing CD8', CD8'ß, or CD8ß-'
chimeric cDNAs. CD8' and CD8'ß transfectants express a splice
variant of CD8 lacking all but four membrane proximal residues of
the CD8 cytoplasmic tail (34, 35). CD8ß-' transfectants express
the extracellular domain of CD8ß together with the transmembrane and
truncated cytoplasmic domains of CD8' in a single chimeric
molecule.
Importantly, addition of SCßDds supernatant did not
significantly affect IL-2 production by parental HTB-157.7 cells
stimulated by L-Kb, excluding SCßDds
interaction with the TCR on HTB-157.7 cells (Fig. 1
A). Similar results
were obtained in the presence or the absence of purified irrelevant mAb
in assays to control for addition of protein or with an equivalent
volume of culture supernatant from cells that did not secrete
SCßDds (data not shown). As expected, all transfectants
expressing CD8, CD8', CD8ß, or CD8'ß exhibited
decreased IL-2 production when SCßDds supernatant was
added (data not shown), consistent with competitive inhibition of the
MHC class I/CD8 interaction. Addition of SCßDds also
led to reduced IL-2 production by CD8ß- and CD8ß-'
transfectants, which express CD8ß extracellular domains in the
absence of surface CD8, as indicated by the percent inhibition of
IL-2 production when stimulated in the presence or the absence of
SCßDds (Fig. 1, B and C).
Although obscured in the compiled results due to variations in low IL-2
values, inhibition of CD8ß- and CD8ß-' transfectants was also
observed at low L-Kb densities, as indicated by the results
of a single representative assay for comparison (Fig. 1C). This pattern of inhibition strongly suggests
that the extracellular domain of CD8ß can interact independently and
directly with MHC class I molecules and/or ß2m in the
absence of CD8. CD8ß-' transfectants displayed somewhat less
pronounced inhibition by SCßDds than did CD8ß-
transfectants (Fig. 1B). It therefore remains
possible that the CD8 cytoplasmic tail affects interaction between
CD8ß and MHC class I molecules and/or ß2m to some
extent.
CD8 interaction with MHC class I governs the response of
CD8ß+ cells to alloantigen
We further investigated CD8 interaction with MHC class I molecules
by stimulating hybridoma transfectants with L cells expressing mutated
MHC class I genes. The mutant-transfectant L cells
(29Kb) express a class I gene harboring a point
mutation at residue 227 (m227) within the 3 domain, which abrogates
binding to CD8 (6, 7). Control stimulator cells (28Kb)
were L cells transfected with otherwise identical MHC class I genes
containing wild-type 3 domains and are capable of interaction with
CD8 (6, 7). We initially compared IL-2 production by individual
transfectants expressing CD8, CD8', CD8ß, CD8'ß,
CD8ß- or CD8ß-' or by untransfected HTB-157.7 cells in
simultaneous assays stimulated by either 28Kb or
29Kb (Fig. 2). Levels of
CD8, CD8ß, and TCRß on the assayed transfectants were analyzed
by cytofluorometry and are shown in Table I.
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, A–G), and the
pattern of responses among these categories was consistent with
previous findings. The observed hierarchy of IL-2 production was:
HTB-157.7 (Fig. 2A) = CD8' (Fig. 2C) < CD8'ß (Fig. 2E) <
CD8 (Fig. 2B) < CD8ß (Fig. 2D). These response differences were most
consistently evident at lower stimulator cell densities, in accord with
previous results (21). In addition, chimeric CD8ß- and CD8ß-'
transfectants (Fig. 2, F–H) produced more IL-2
than untransfected cells in response to 28Kb (Fig. 2A), and this level was lower than that produced by
similarly stimulated CD8 transfectants (Fig.
2B). This increase relative to untransfected
hybridoma cells is not convincingly evident in Figure 2 and may be
explained by the very low levels of transfected gene product expressed
on the surface of the CD8ß- and CD8ß-' transfectants used in
this particular assay (see Table I).
Parental HTB-157.7, CD8ß-, and CD8ß-' transfectants all
responded better to 29Kb than to 28Kb
stimulators (Fig. 2, A and E–G) in the
assay shown. In contrast, CD8, CD8', and CD8ß transfectants
all responded poorly to 29Kb relative to 28Kb
stimulators (Fig. 2, B–D). The level of IL-2
production in response 29Kb relative to 28Kb
varied somewhat from assay to assay, probably due to fluctuating levels
of transfected MHC class I products on the two stimulator cell lines
(such fluctuation has been observed on several of our MHC class
I-transfected L cell lines (C. J. Wheeler, unpublished
observations)). However, the same pattern of response among the
different categories of transfectants was observed in every assay (data
not shown). The observed pattern suggests that the responses of
CD8+ or CD8ß+ hybridoma cells are
dependent upon interaction between CD8 and MHC class I 3 domains.
Thus, the question of CD8ß’s capacity for independent interaction
with MHC class I molecules in a more physiologic context is unresolved
by these data. However, it is clear that extracellular CD8
expression is overriding in its ability to mediate decreased responses
to 29Kb relative to 28Kb, and coexpression of
CD8ß does not alter this property.
More revealing was the observation that CD8'ß transfectants
exhibited a mixed pattern of relative responses to 29Kb and
28Kb. Some CD8'ß transfectants (Fig. 2E,
a'b.4) displayed minimal diminution of IL-2 production in response to
29Kb relative to 28Kb stimulators (similar to
untransfected and CD8ß- and CD8ß-' transfectants), whereas
others (Fig. 2E, a'b.5) exhibited relatively decreased
responses to 29Kb stimulators (similar to CD8, CD8',
and CD8ß transfectants). This suggests that CD8ß expression may
decrease the dependence of the IL-2 response on interaction between
extracellular CD8 and MHC class I in the absence of a CD8
cytoplasmic tail. Analysis of additional CD8'ß transfectants
indicated that this decreased dependence was somewhat encouraged by
higher levels of CD3 on transfectants, by low ratios of surface
CD8 to CD8ß molecules on transfectants, or by both (Fig. 2, Table I). Thus, a property of the intact CD8 cytoplasmic tail might
obscure CD8ß-mediated response enhancement, and expression of a
cytoplasmic tailless form of CD8 (CD8') can reveal this effect.
Taken together, these data indicate that CD8ß can independently
interact with MHC class I/ß2m on target cells, but that
such interaction is secondary to and dependent upon initial
interaction between wild-type CD8 and MHC class I 3 domains.
CD8 inhibits and CD8ß restores response to anti-CD3
mAb
To assess whether CD8ß is able to enhance TCR-mediated
responses in the absence of MHC class I, we stimulated hybridoma
transfectants with plate-bound mAb to CD3. Transfectants were
analyzed for levels of surface CD8, CD8ß, and CD3 by
cytofluorometry before assay (see Table II). Compiled results for untransfected
hybridomas (HTB), CD8, CD8ß, and CD8'ß transfectants are
shown in Figure 3. Since the compiled
results are subject to substantial variation in values obtained from
assays performed at different times, not all consistent
trends are necessarily apparent for all densities of
L-Kb stimulators. For this reason, results
representative of eight independent experiments from individual
transfectants in a single assay are also shown in Figure 4. Transfectants that expressed CD8
alone (Fig. 3, A–C, and Fig. 4A)
produced significantly decreased levels of IL-2 compared with
untransfected hybridoma cells (Figs. 3A and
4A). In contrast, coexpression of CD8
and CD8ß or CD8' and CD8ß on responding cells did not result in
inhibition of IL-2 production relative to that of untransfected cells
under identical stimulation conditions (Fig. 3, A–C, and
Fig. 4A).
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transfectants were
generally defective or that CD8ß, CD8'ß, or untransfected
cells were generally superior in their ability to respond to TCR
stimulation, we conducted assays in which identical transfectants were
stimulated in parallel with anti-CD3 mAb or L-Kb.
CD8ß and CD8'ß transfectants exhibited very robust IL-2
production in response to both anti-CD3 mAb and L-Kb
stimulation (Fig. 4B). In contrast, CD8
transfectants produced relatively more IL-2 in response to
L-Kb than to anti-CD3 mAb (Fig. 4C and
data not shown). This indicates that the enhanced responsiveness to
anti-CD3 mAb did not simply reflect a greater ability of
CD8ß+ transfectants or untransfected cells to respond in
general. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or CD8ß-' chimeric molecules)
suggests that CD8ß is capable of direct independent interaction with
MHC class I/ß2-microglobulin. The patterns of IL-2
production by transfectants expressing different isoforms of CD8 in
response to MHC class I alloantigen (m227) unable to interact with
CD8 suggest that CD8ß interaction with MHC class I may require
initial interaction between CD8 and MHC class I 3 domains. In
addition, CD8'ß transfectants exhibited mixed responses to m227,
suggesting that CD8ß-mediated response enhancement may be influenced
by the cytoplasmic domain of CD8 or factors associated with it in a
manner related to the CD8/ß ratio, the CD3 level, or both.
Finally, CD8, but not CD8ß or CD8'ß, inhibited responses
to direct stimulation of the TCR/CD3 complex in the absence of external
alloantigenic MHC class I, suggesting that CD8ß influences the
ability of CD8 to functionally couple to TCR/CD3. These results expand
upon recent findings that CD8ß more avidly strengthens
the TCR-ligand interaction than does CD8 (36).
The responses of untransfected HTB-157.7 cells were not inhibited by
addition of SCßDds, whereas those of CD8ß- and
CD8ß-' transfectants were. Since all these responses were
relatively unaffected by m227, these data indicate that interaction
between CD8ß and MHC does not critically involve residue 227 in the
class I 3 domain. A previous study compared responses of
CD8+ and CD8ß+ hybridoma transfectants
to stimulators expressing MHC class I molecules unable to interact
efficiently with CD8 (22). This report suggested that CD8ß
molecules bind to MHC class I 3 domains in much the same manner as
homodimeric CD8, albeit with somewhat enhanced avidity. Our results
are not inconsistent with these conclusions, but indicate additional
restrictions for CDß interaction with MHC class
I/ß2m.
Since SCßDds was able to effectively compete for binding
by CD8ß- molecules to H-2Kb on stimulator L cells,
residues common to at least H-2Dd (from which
SCßDds is derived) and H-2Kb (transfected
class I gene product in L cell stimulators) and/or residues on
ß2m may interact with the extracellular domain of CD8ß.
Thus, nonunique residues on class I heavy chains and/or
ß2m may govern the interaction with CD8ß.
It is difficult to assess the relative strength of CD8 and CD8ß
interactions with MHC class I and/or ß2m because the
transfectants that express CD8 and CD8ß independently (CD8 or
CD8' and CD8ß- or CD8ß-' transfectants, respectively)
express widely different levels of transfected gene products. It may be
possible to measure the strengths of these respective interactions by
analyzing the affinities of purified CD8, CD8', CD8ß-, or
CD8ß-' molecules for SCßDds using surface plasmon
resonance. However, our data indicate that interactions between either
CD8 or CD8ß and MHC class I/ß2m contribute
measurably to IL-2 production by HTB-157.7 transfectants.
Hybridoma transfectants expressing no CD8 (including chimeric
CD8ß- or CD8ß-' transfectants) responded at least as well to
mutant class I (29Kb) unable to interact with CD8 as to
control class I (28Kb) permissive for this interaction. In
contrast, transfectants expressing CD8, CD8ß, or CD8'
responded relatively poorly to 29Kb. This suggests that
interaction between CD8 and MHC class I governs responsiveness, and
expression of CD8ß does nothing to override this requirement. In
contrast, some CD8'ß transfectants consistently responded as well
or better to 29Kb relative to controls, whereas CD8'
transfectants responded relatively poorly to 29Kb. It could
be argued that this effect is related to changes in the stoichiometry
of Lck and the TCR/CD3 complex, such that greater association of Lck
with the TCR/CD3 complex and enhanced TCR signaling to
29Kb, in turn, might occur in the absence of the CD8
cytoplasmic tail. However, preferential influences on responses to
29Kb are difficult to incorporate into such a model.
Moreover, the absence of the CD8 cytoplasmic tail in CD8'ß
relative to CD8ß transfectants did not lead to alteration of
responses to anti-CD3 mAb (Fig. 3). This suggests that CD8ß
directly affects response enhancement to both alloantigen and
anti-CD3 mAb when associated with cytoplasmic tailless
CD8.
CD8 had an inhibitory effect on IL-2 production only when stimulated
with anti-CD3 mAb, and CD8ß coexpression relieved this
CD8-mediated inhibition. An inhibitory effect on TCR-mediated
responses has also been observed upon ligation of CD4 (37). In the
latter case, inhibition was ascribed to sequestration of Lck away from
the TCR complex by CD4 ligation. Given the relative independence from
CD3 levels (compare, for example, a.1 with ab.1 in Table II), we
think that this effect cannot be explained solely by quantitative
differences in Lck associated with CD8 and CD8ß dimers. In
fact, it has been shown that CD8ß associates with significantly
more Lck kinase activity than does CD8 (24). Since
CD8ß+ cells respond more robustly to alloantigen as
well, we believe that the CD8ß-mediated relief of inhibition in mAb
stimulation assays reflects a unique ability to mediate efficient
association with TCR/CD3. A similar association between CD4 and the TCR
has been reported (38, 39). We have also observed CD3 in Western
blots of anti-CD8 immunoprecipitates from activated transfectants
that express extracellular CD8ß (data not shown), but not from those
expressing only CD8. This supports the idea that CD8ß mediates
physical as well as functional association with the TCR/CD3
complex.
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Footnotes |
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2 Current address: Cedars-Sinai Neurosurgical Institute, 8635 W. Third Street, Los Angeles, CA 90048.
3 Address correspondence and reprint requests to Dr. Jane R. Parnes, Division of Immunology and Rheumatology, MSLS P-306, Stanford University Medical Center, Stanford, CA 94305-5487.
Received for publication September 11, 1997. Accepted for publication December 29, 1997.
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(Ly-2) subunit on activated thymic / cells. Eur. J. Immunol. 20:927.[Medline]
3 domain of an H-2 class I molecule abrogates reactivity with CTL. J. Exp. Med. 166:956.3 domain: CD8 and the T-cell receptor recognize the same class I molecule. Proc. Natl. Acad. Sci. USA 87:2137.3 domain of HLA-A2. Nature 345:41.[Medline]
chain-associated Lck kinase activity. J. Exp. Med. 181:1267.' polypeptides to associate with Lck correlates with impaired function in vitro and lack of expression in vivo. Nature 342:278.[Medline]
ß T-cell receptor. Nature 358:328.[Medline]
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