By
From the * Basel Institute for Immunology, 4005, Basel, Switzerland; and Molecular Immunology
Unit Institut Pasteur, 75724 Paris Cedex 15, France
CD4 and CD8 are thought to function as coreceptors by binding to the cognate major histocompatibility complex (MHC) molecules recognized by the T cell antigen receptor (TCR) and initiating the signal transduction cascade. We report that during T cell-antigen-presenting cell interaction, triggered TCRs and coreceptors are downregulated and degraded with identical kinetics. This coordinated disappearance takes place whenever the TCR is triggered, even when the coreceptor does not engage the cognate MHC molecule and is the consequence of binding of the coreceptor-associated Lck to ZAP-70. The interaction of coreceptor and cognate MHC molecules is dispensable when T cells are stimulated by optimal ligands, but becomes crucial when suboptimal ligands are used. In the latter case the coreceptor increases the efficiency of TCR triggering without changing the activation threshold or the quality of the T cell response.
Because of their capacity to bind to the same MHC
molecule as that engaged by the TCR (1, 2), and because of their association with the tyrosine kinase Lck (3),
CD4 and CD8 have been defined as coreceptors (4, 5). The
binding of coreceptor to the cognate MHC molecule is
thought to perform two functions: (a) to stabilize the TCR-
peptide MHC interaction (6); and (b) to carry Lck in contact with the TCR to initiate phosphorylation events (9).
Indeed, cross-linking of TCRs and coreceptors results in
enhanced T cell response (10, 11), whereas interference with
the coreceptor-MHC interaction inhibits T cell activation or changes the quality of the response (12).
However, there are clear cases where the interaction of
coreceptors with cognate MHC molecules appears to be
dispensable for full T cell activation (15). These findings raise
the question of whether the contribution of the coreceptor
to T cell activation is qualitative, in the sense that it provides signals additional to and different from those provided
by TCR alone, or whether the coreceptor acts by facilitating the triggering of TCRs when the interaction has lower
than optimal kinetics.
We report that during T cell-APC interaction, the coreceptors are recruited to triggered TCRs and are downregulated with identical kinetics, even when the coreceptor
does not engage the cognate MHC molecule. This process
is the consequence of the binding of coreceptor-associated
Lck to ZAP-70/ T Cell Clones.
HLA-DR1101-restricted (KS140, KS70, and
KS164) and DR1302-restricted (AL15.1) CD4+ T cell clones
specific for tetanus toxin (TT)830-843 peptide and HLA-A2-
restricted CD8+clones (CER22, CER43) specific for the influenza
matrix (M) 58-66 peptide were used. T cell clones were conjugated with EBV-transformed B (EBV-B) cells pulsed with various
concentrations of either peptide or bacterial superantigens (toxic
shock syndrome toxin or staphylococcal enterotoxin B) or monovalent anti-CD3 antibodies (W632/T3 or L243/T3) as described
(16). CD4+CD8+ alloreactive T cell clones were isolated by sorting
double positive cells from a primary MLR. CD8+ alloreactive T
cell clones that recognize class II molecules on the class I FACS® Analysis.
T cells were conjugated with autologous
EBV-B cells pulsed with peptide, superantigen, or anti-CD3 for 5 h
at 37°C. Downregulation and degradation of TCR/CD3 and
coreceptors were measured by indirect immunofluorescence on
intact and permeabilized cells as previously described (17, 18) using antibodies to CD3 (OKT3 or Leu3a), CD4 (6D10), and CD8
(OKT8). The absolute numbers of CD3, CD4, and CD8 molecules per cell were estimated by reference to a standard curve of
beads coated with known amounts of mouse Ig (Flow Cytometry Standards Europe, Leiden, The Netherlands).
Immunoprecipitations and Kinase Assays.
T cells were stimulated or not with the appropriate APC for 2 min at 37°C and lysed for 30 min at 4°C in 1% Brij96 buffer (20 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1 mM MgCl2, and 1 mM EGTA) in the
presence of protease and phosphatase inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM Pefabloc-SC, 50 mM NaF, 10 mM
Na4P2O7, and 1 mM NaVO4). CD4 immunoprecipitation, in
vitro kinase assay, and reimmunoprecipitation with anti-ZAP-70
or anti- Drugs and Transfection.
Genistein and herbimycin A were
purchased from Calbiochem (La Jolla, CA) and used as previously
described (21). After overnight treatment with herbimycin, which is
required to deplete Lck, the expression of coreceptor was reduced
by ~50%. cDNAs encoding wild-type human CD4 and the mutant CD4.401 molecule that does not associate with Lck were a
gift of Dr. Dan Littman (Skirball Institute of Biomolecular Medicine, NYU Medical Center, New York; reference 22). The cDNA
was subcloned in the pSAM-EN retroviral vector (23) using the
SalI-XhoI restriction sites. Plasmid DNA was purified and transfected in the amphotropic packaging cell line PA317 as previously
described (22). The transduction of Jurkat cells was done as previously described (22).
Substituted Peptides and Anticoreceptor Antibodies.
Several substituted analogs of TT830-843 were tested for their capacity to
trigger T cell proliferation, and peptides with unaltered capacity
to bind to DR molecules but weaker T cell stimulatory capacity
were selected. Blocking anti-CD4 antibodies (M-T310, M-T413,
M-T414, and M-T435) and anti-CD8 (733) were a gift of Dr.
E.P. Rieber (Institute of Immunology, Technical University Dresden, Germany) and Dr. E. Roosnek (University Hospital, Geneva, Switzerland). Antibodies were used in the culture at 10 µg/ml.
To understand the contribution of the coreceptor to TCR triggering and T cell activation, we studied
the TCR-coreceptor interaction in T cells stimulated by a
specific ligand. It has been shown that, after triggering by
agonists, TCRs are downregulated and degraded and this
downregulation can be used to measure the number of
TCRs triggered (17, 18). Therefore, we investigated whether the CD4 or CD8 coreceptors would also be downregulated
together with the TCR. We observed that in specific T-APC
conjugates, TCRs and coreceptors are downregulated with
the same kinetics and with fixed stoichiometry (Fig. 1, A and
B). In addition, downregulation is followed by rapid degradation of both coreceptor and TCR (Fig. 1 C). By reference to a standard curve of Ig-coated beads, we estimated
that approximately two CD4 or four CD8 molecules are downregulated for each TCR (data not shown), an estimate which is consistent with that reported for CD4 by
Saizawa and Janeway (24).
We investigated whether the codownregulation of TCR and coreceptor would require the interaction
between coreceptor and cognate MHC molecule or whether
it would simply be a consequence of TCR triggering.
Three lines of evidence indicate that coreceptor downregulation can occur in the absence of interaction with the
cognate MHC molecule. First, a parallel downregulation of
TCR and coreceptor was induced not only by specific
peptide-MHC complexes, but also by bacterial superantigens or monovalent anti-CD3 antibodies (Fig. 2, A and B).
Second, class I-restricted T cell clones expressing both CD4 and CD8 downregulated both coreceptors to the same extent (Fig. 2 C). Third, class II-alloreactive CD8+ T cell
clones, when stimulated by class II+ APCs lacking class I
molecules, downregulated CD8 together with TCR (Fig.
2 D).
The mechanism responsible for the downregulation of
the coreceptor was next investigated. Three lines of evidence indicate that the downregulation of the coreceptor
was due to an intracellular association with triggered TCRs.
First, immunoprecipitation experiments showed that, as
previously demonstrated in Jurkat cells activated by anti-CD3 antibodies (19, 20), a complex-containing coreceptor, Lck and ZAP-70, is formed after T cell activation by a specific ligand (Fig. 3 A). Comparable results were obtained
when the reimmunoprecipitation was performed with either anti-ZAP-70 or anti-
Taken together, the above results
demonstrate that coreceptors become associated to, and are
downregulated together with, triggered TCRs, and that
this process can occur after TCR triggering, even in the
absence of interaction of the coreceptor with the cognate
MHC molecules. However, these results do not explain how coreceptors contribute to T cell antigen recognition in
most cases. Considering the serial engagement model (25),
it is conceivable that the requirement for coreceptor might
vary with the kinetics of the TCR-ligand interaction. Although ligands with optimal kinetics might efficiently trigger TCRs even in the absence of coreceptor, ligands with
higher off-rates may require the extracellular interaction of
the coreceptor with the cognate molecule to increase the
stability of the complex and, consequently, the rate of triggering.
To address this point, we stimulated T cell clones with
ligands of different potency and tested the effect of CD4 or
CD8 antibodies on the extent of TCR triggering, the T cell
activation threshold, and the quality of the response. As
shown in Fig. 4, the proliferative response of T cell clone
KS70 was not affected by anti-CD4 antibody when the clone
was triggered by the bacterial superantigen TSST, but
was inhibited by anti-CD4 when the clone was triggered
by peptide-MHC complexes. Interestingly, clone KS164,
which displayed a better dose-response curve to the same
peptide-DR complex, was not inhibited by anti-CD4. However, this clone became sensitive to inhibition when a modified peptide with weaker agonistic properties was used. Comparable results were obtained with two additional CD4+
and two additional CD8+ clones, indicating that the sensitivity to inhibition by anticoreceptor antibodies is inversely
correlated to the efficiency of the TCR-ligand interaction.
To investigate whether the lower response of T cells in
the presence of anti-CD4 antibodies reflects a lower extent
of TCR triggering or, rather, an altered signal leading to
qualitatively different responses, we correlated TCR downregulation and cytokine production in T cells stimulated by
strong or weak agonists in the presence or absence of anti-CD4 antibodies. As shown in Fig. 5, the inhibition of T
cell response by anti-CD4 antibodies precisely correlated
with a reduced level of TCR downregulation both when strong and weak agonists were used. These results indicate
that the anti-CD4 antibody mimics the effect of a weak agonist, i.e., results in a decreased efficiency of serial triggering. However, in spite of a decreased efficiency of TCR
triggering, the threshold of T cell activation and the type of
cytokines produced were not affected by anti-CD4. Indeed, T cells produced IFN-
Our results reconcile several apparently contradictory
observations concerning coreceptor dependency and the role
of extracellular and intracellular interactions in coreceptor
function. We have shown that in T cells activated by peptide-MHC, superantigens, or anti-CD3 antibodies the coreceptors are recruited to triggered TCRs and are downregulated and degraded together with them. This process
does not necessarily require the interaction of the coreceptor with the cognate MHC molecule, but takes place
whenever the TCR is triggered via the intracellular association of Lck and ZAP-70/ It is interesting that even in unstimulated T cell clones There is clear evidence that coreceptor can stabilize the
TCR/peptide-MHC interaction (6). We have shown here
that this stabilization may actually result in an increased rate
of TCR triggering. Indeed, the coreceptor appears to be
dispensable when the ligands have optimal kinetics, allowing efficient serial TCR triggering. However, the binding
of coreceptor to cognate MHC molecules becomes critical
in the case of low-affinity ligands because in this case the
coreceptor can initially stabilize the TCR-ligand interaction, thus increasing the probability that an engagement
event will result in triggering.
Our results also show that coreceptors play a quantitative
role in T cell activation. Indeed the reduced response observed in cultures stimulated with suboptimal ligands or in
the presence of anticoreceptor antibodies can be fully accounted for by a reduced level of TCR triggering. The fact
that the T cell activation thresholds and the profile of cytokines produced are comparable in all conditions of stimulation is not surprising. Indeed, in all cases the triggered TCRs have been shown to be complexed with coreceptors, suggesting similar composition and transduction capacity of the signaling complexes.
and takes place whenever the TCR is
triggered. We also show that the contribution of the coreceptors becomes crucial when suboptimal ligands are used. In this case, engagement of the coreceptor with the cognate MHC molecule increases the efficiency of serial TCR
triggering without changing the activation threshold or the
quality of the T cell response.
EBV-B
.221 cells were generated, stimulating PBMC with irradiated .221 cells, followed by sorting and cloning of the CD8+CD4
cells.
The class II specificity of the clones was verified using a panel of
typed EBV-B cells as well as inhibition by anti-class II antibodies.
were performed as previously described (19, 20) using
the following antibodies: 19Thy-5D7 (IgG2a, anti-CD4), 21Thy-2D3 (IgG1, anti-CD8), and rabbit polyclonal antibodies to ZAP-70 and
.
Parallel Downregulation and Degradation of the Coreceptor
and TCR.
Fig. 1.
Parallel downregulation
and degradation of CD4 and CD8 together with CD3 in T cell clones stimulated by specific antigen. (A) Time
course of CD3 () and CD4 (
) downregulation in clone KS140 stimulated
with APCs pulsed with 10 nM TT830-842. (B) CD3 (
) and CD8 (
) downregulation in clone CER22 stimulated
with APCs pulsed with 100 nM M58-66. (C) Surface and total levels of CD3 and CD4 as determined by staining before and after permeabilization in KS70 cells conjugated for 5 h with APCs
unpulsed (empty bars) or pulsed with 0.1 µM (hatched bars) or 10 µM (filled bars) TT830-842.
[View Larger Versions of these Images (12 + 23K GIF file)]
Fig. 2.
Downregulation of coreceptors can occur in the absence of
an interaction with cognate or noncognate MHC molecules. Downregulation of CD3 and coreceptor in CD4+ (KS70; A) or CD8+ (CER43; B)
T cell clones stimulated by specific peptide-MHC (), superantigens
(
), or monovalent anti-CD3 antibodies: w632/T3 (
), L243/T3 (
).
(C) Downregulation of CD3 versus CD4 (
) and CD8 (
) in alloreactive CD4+ CD8+ T cell clones stimulated by specific alloantigen (D).
Downregulation of CD3 and CD8 in CD8+, class II-alloreactive T cell
clones stimulated with class I
APCs (.221) expressing the relevant class II
alloantigen.
[View Larger Version of this Image (27K GIF file)]
. Second, treatment with the
Lck inhibitors genistein or herbimycin A does not interfere with TCR downregulation, but completely inhibits coreceptor downregulation (data not shown). Third, truncated
CD4 molecules that fail to associate with Lck are not
downregulated with triggered TCRs (Fig. 3 B).
Fig. 3.
Intracellular association of coreceptor with triggered
TCRs. (A) T cell clones were
stimulated (+) or not () with
APCs, lysed, and the CD4 or
CD8 immunoprecipitates were
subjected to an in vitro kinase assay, followed by reimmunoprecipitation with anti-ZAP-70 antiserum. CD4+ clone KS140
conjugated with peptide-pulsed
or unpulsed APCs (lanes 1 and
2). CD8+ clone MS3 either untreated or conjugated with allogeneic class I
APC (lanes 3 and 4). (B)
Downregulation of CD3 (
,
) and CD4 (
,
) in CD4
Jurkat cells
transfected with wild-type CD4 (
,
) or with mutant CD4.401 (
,
).
[View Larger Versions of these Images (20 + 20K GIF file)]
Fig. 4.
The sensitivity to
inhibition by anti-CD4 depends
on the nature of the ligand. (A
and B) Proliferative response of
clone KS70 to various doses of
TSST or TT830-843 in the
presence () or absence (
) of
anti-CD4. (C) Proliferative response of clone KS164 to TT830-843 (
,
) or to
TT830-843 Ala839-substituted
peptide that behaves as a weak
agonist (
,
) in the presence
(
,
) or absence (
,
) of
anti-CD4.
[View Larger Version of this Image (13K GIF file)]
, IL-2, and TNF-
when
~20-30% of TCRs were triggered, irrespective of the strength of the agonist and of the presence or absence of
anti-CD4. IL-3 production consistently required higher
levels of TCR occupancy, which were comparable in all
experimental conditions.
Fig. 5.
Quantitative contribution of CD4 to TCR triggering and T cell activation. (A)
IFN- production by T cell
clone AL15.1 stimulated by
TT830-843 (
,
) or by
TT830-843 Gln839 (
,
) in the
presence (
,
) or absence (
,
) of anti-CD4 antibody. (B)
CD3 downregulation in the
same experiment. (C-E) Levels
of IFN-
(
), IL-2 (
), TNF-
(
), and IL-3 (
) as a function
of the number of TCRs downregulated in cultures stimulated
by wild-type agonist (C), wild-type agonist + anti-CD4 (D), or
weak agonist (E). No cytokine
production was observed in cultures stimulated by weak agonist + anti-CD4.
[View Larger Version of this Image (25K GIF file)]
.
and ZAP-70 can be immunoprecipitated by anti-CD4 or
anti-CD8 antibodies, although the complexes contain only
low levels of kinase activity. This finding suggests that in
human as well as mouse T cells a fraction of TCRs is constitutively associated with the coreceptor (26). This association may be responsible for the constitutive association of
phospho-
and ZAP-70 observed in thymocytes and lymph
node T cells (27). It is tempting to speculate that the constitutive association of TCR and coreceptors might play a
role in inducing positive selection by self-ligands in the
thymus as well as in facilitating the response to low-affinity
ligands in periphery.
Address correspondence to Antonella Viola, Basel Institute for Immunology, Grenzacherstrasse 487, CH4005, Basel, Switzerland. Phone: 41-61-605-1210; FAX: 41-61-605-1222; E-mail: viola{at}bii.ch
Received for publication 9 July 1997 and in revised form 12 September 1997.
The Basel Institute for Immunology was founded and is supported by F. Hoffmann La Roche Ltd., Basel, Switzerland.We thank E.P. Rieber and E. Roosnek for providing blocking anti-CD4 and anti-CD8 antibodies, D. Littmann for providing CD4 constructs, and K. Karjalainen and M. Cella for critical reading and comments.
1. | Doyle, C., and J.L. Strominger. 1987. Interaction between CD4 and class II MHC molecules mediated cell adhesion. Nature (Lond.). 330: 256-259 [Medline]. |
2. | Norment, A.M., R.D. Salter, P. Parham, V.H. Engelhard, and D.R. Littman. 1988. Cell-cell adhesion mediated by CD8 and MHC class I molecules. Nature (Lond.). 336: 79-81 [Medline]. |
3. | Veillette, A., M.A. Bookman, E.M. Horak, and J.B. Bolen. 1988. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 55: 301-308 [Medline]. |
4. | Janeway, C.A. Jr.. 1988. T cell development. Accessories or coreceptors? Nature (Lond.). 335: 208-210 [Medline]. |
5. | Janeway, C.A. Jr.. 1991. The co-receptor function of CD4. Semin. Immunol. 3: 153-160 [Medline]. |
6. | Luescher, I.F., E. Vivier, A. Layer, J. Mahiou, F. Godeau, B. Malissen, and P. Romero. 1995. CD8 modulation of T cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes. Nature (Lond.). 373: 353-356 [Medline]. |
7. |
Renard, V.,
P. Romero,
E. Vivier,
B. Malissen, and
I.F. Luescher.
1996.
CD8![]() |
8. | Garcia, K.C., C.A. Scott, A. Brunmark, F.R. Carbone, P.A. Peterson, I.A. Wilson, and L. Teyton. 1996. CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes [see comments]. Nature (Lond.). 384: 577-581 [Medline]. |
9. | Weiss, A., and D.R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. Cell. 76: 263-274 [Medline]. |
10. | Emmrich, F., U. Strittmatter, and K. Eichmann. 1986. Synergism in the activation of human CD8 T cells by cross-linking the T-cell receptor complex with the CD8 differentiation antigen. Proc. Natl. Acad. Sci. USA. 83: 8298-8302 [Abstract]. |
11. | Janeway, C.A. Jr., and K. Bottomly. 1994. Signals and signs for lymphocyte responses. Cell. 76: 275-285 [Medline]. |
12. |
Madrenas, J.,
L.A. Chau,
J. Smith,
J.A. Bluestone, and
R.N. Germain.
1997.
The efficiency of CD4 recruitment to
ligand-engaged TCR controls the agonist/partial agonist
properties of peptide-MHC molecule ligands.
J. Exp. Med.
185:
219-229
|
13. | Vidal, K., B.L. Hsu, C.B. Williams, and P.M. Allen. 1996. Endogenous altered peptide ligands can affect peripheral T cell responses. J. Exp. Med. 183: 1311-1321 [Abstract]. |
14. |
Mannie, M.D.,
J.M. Rosser, and
G.A. White.
1995.
Autologous rat myelin basic protein is a partial agonist that is converted into a full antagonist upon blockade of CD4. Evidence
for the integration of efficacious and nonefficacious signals
during T cell antigen recognition.
J. Immunol.
154:
2642-2654
|
15. | Cerundolo, V., A.G. Tse, R.D. Salter, P. Parham, and A. Townsend. 1991. CD8 independence and specificity of cytotoxic T lymphocytes restricted by HLA-Aw68.1. Proc. R. Soc. Lond. B. Biol. Sci. 244: 169-177 [Medline]. |
16. | Viola, A., and A. Lanzavecchia. 1996. T cell activation determined by T cell receptor number and tunable thresholds [see comments]. Science (Wash. DC). 273: 104-106 [Abstract]. |
17. | Valitutti, S., S. Muller, M. Cella, E. Padovan, and A. Lanzavecchia. 1995. Serial triggering of many T cell receptors by a few peptide-MHC complexes [see comments]. Nature (Lond.). 375: 148-151 [Medline]. |
18. |
Valitutti, S.,
S. Muller,
M. Salio, and
A. Lanzavecchia.
1997.
Degradation of T cell receptor (TCR)-CD3-![]() |
19. |
Thome, M.,
P. Duplay,
M. Guttinger, and
O. Acuto.
1995.
Syk and ZAP-70 mediate recruitment of p56lck/CD4 to the
activated T cell receptor-CD3/![]() |
20. |
Thome, M.,
V. Germain,
J.P. DiSanto, and
O. Acuto.
1996.
The p56lck SH2 domain mediates recruitment of CD8/
p56lck to the activated T cell receptor/CD3/![]() |
21. | Salio, M., S. Valitutti, and A. Lanzavecchia. 1997. Agonist-induced TCR downregulation: molecular requirements and dissociation from T cell activation. Eur. J. Immunol. 27: 1769-1773 [Medline]. |
22. | Bedinger, P., A. Moriarty, R.C. von Borstel II, N.J. Donovan, K.S. Steimer, and D.R. Littman. 1988. Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4. Nature (Lond.). 334: 162-165 [Medline]. |
23. | Candotti, F., S.A. Oakes, J.A. Johnston, L.D. Notarangelo, J.J. O'Shea, and R.M. Blaese. 1996. In vitro correction of JAK3-deficient severe combined immunodeficiency by retroviral-mediated gene transduction. J. Exp. Med. 183: 2687-2692 [Abstract]. |
24. |
Saizawa, K., and
C.A. Janeway Jr..
1987.
Evidence for a
physical association of CD4 and the CD3:![]() ![]() |
25. | Valitutti, S., and A. Lanzavecchia. 1997. Serial triggering of T-cell receptors: a basis for the sensitivity and specificity of T cell antigen recognition. Immunol. Today. 18: 299-304 [Medline]. |
26. |
Diez-Orejas, R.,
S. Ballester,
M.J. Feito,
G. Ojeda,
G. Criado,
M. Ronda,
P. Portoles, and
J.M. Rojo.
1994.
Genetic
and immunochemical evidence for CD4-dependent association of p56lck with the ![]() ![]() |
27. |
van Oers, N.S.C.,
N. Killeen, and
A. Weiss.
1994.
ZAP-70 is
constitutively associated with tyrosine-phosphorylated TCR
![]() |