From the Laboratory of Immunobiology, Dana-Farber Cancer Institute and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 27, 2000, and in revised form, December 18, 2000
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ABSTRACT |
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CD2 mediates T cell adhesion via its
ectodomain and signal transduction utilizing its 117-amino acid
cytoplasmic tail. Here we show that a significant fraction of human CD2
molecules is inducibly recruited into lipid rafts upon CD2
cross-linking by a specific pair of mitogenic anti-CD2 monoclonal
antibodies (anti-T112 + anti-T113) or
during cellular conjugate formation by CD58, the physiologic ligand
expressed on antigen-presenting cells. Translocation to lipid
microdomains is independent of the T cell receptor (TCR) and, unlike
inducible TCR-raft association, requires no tyrosine phosphorylation. Structural integrity of rafts is necessary for CD2-stimulated elevation of intracellular free calcium and tyrosine phosphorylation of cellular substrates. Whereas murine CD2 contains two
membrane-proximal intracellular cysteines, partitioning CD2 into
cholesterol-rich lipid rafts constitutively, human CD2 has no
cytoplasmic cysteines. Mapping studies using CD2 point mutation, deletion, and chimeric molecules suggest that conformational change in
the CD2 ectodomain participates in inducible raft association and
excludes the membrane-proximal N-linked glycans, the
transmembrane segment, and the CD2 cytoplasmic region (residues 8-117)
as necessary for translocation. Translocation of CD2 into lipid
rafts may reorganize the membrane into an activation-ready state
prior to TCR engagement by a peptide associated with a major
histocompatibility complex molecule, accounting for synergistic T cell
stimulation by CD2 and the TCR.
T cell activation occurs following binding of T cell receptors
(TCRs)1 to peptides
associated with major histocompatibility complex molecules (pMHC). This
process is highly specific and sensitive despite the fact that the
binding affinity between the TCR and pMHC complex is low, with
relatively fast off-rates (1). Moreover, a small number of pMHC per
antigen-presenting cell (APC) is sufficient to trigger and activate T
cells (2). To maintain such a high degree of sensitivity and fidelity
of T cell recognition, a number of co-stimulatory and accessory T cell
surface molecules are coordinately engaged in the immune recognition
process. These include CD2, CD4, CD8, CD28, and LFA-1 (3-6) among
others. Furthermore, activation signals are induced, whereas signals
inhibiting cell growth and effector function are suppressed.
Recently, plasma membrane compartmentation was shown to be involved in
TCR signaling and the partitioning of essential T cell-activating components (reviewed in Refs. 7-11). Certain constituents of the plasma membrane domain form an ordered liquid phase with reduced membrane fluidity (12), and these are termed lipid rafts,
glycolipid-enriched membrane domains, or detergent-insoluble
glycolipid-enriched microdomains (DIGs). These rafts maintain a
distinct composition of lipid, sphingolipid, and cholesterol (12, 13)
and are resistant to mild detergent extraction, floating to the low
density fraction on sucrose gradients (7, 10), and can be easily
isolated. They are thought to participate in protein sorting, membrane
trafficking, and signal transduction (10) since numerous signaling
molecules were found localized in DIGs (7, 10). These include Src
family protein-tyrosine kinases (14, 15), heterotrimeric and monomeric Ras-like G proteins (16, 17), molecules involved in Ca2+
flux (18), as well as the small signaling molecule phosphatidylinositol bisphosphate (17, 19). The co-localization of these signaling molecules
with lipid rafts is largely due to post-translational acylation
modification of proteins (7), the most important being
membrane-proximal cysteine residue palmitoylation (reviewed in Ref.
20). Another post-translational modification that can bring cell
surface proteins to the lipid rafts is glycosylphosphatidylinositol modification (21). Representatives of this class of cell surface molecule include Thy-1, CD48, CD58, Ly-6 ,and CD24. Although these structures lack both cytoplasmic and transmembrane domains, their cross-linking by suitable ectodomain-specific antibodies results in
signal transduction and cellular responses.
The idea that lipid rafts can serve as a platform for lymphocyte
signaling is supported by several recent reports demonstrating that
multiple-chain immunoreceptors such as TCRs (17, 22, 23), B cell
receptors (24, 25), and high affinity IgE receptors (Fc The transmembrane T cell surface protein CD2 molecule is expressed on
virtually all T cells, thymocytes, and natural killer cells (6).
Human CD2 promotes the physical interaction of T and natural killer
lineage cells with APCs, stromal elements, and a variety of target
cells bearing the ubiquitously expressed CD2 ligand, CD58 (3, 6, 30,
31). CD2 functions to mediate T cell adhesion as well as to facilitate
signal transduction. CD2 initiates T cell-APC contact even prior to TCR
recognition of the pMHC (6, 32, 33). The ligation of CD2 by CD58 not only promotes the adhesion of T cells to APCs, but provides a suitable
intercellular membrane spacing (~140 Å) for TCR-pMHC recognition (34). Furthermore, the extremely fast on- and off-rates of
the CD2-CD58 interaction (35) enable a TCR to scan APC surfaces searching for the specific agonistic pMHC. Although the monomeric CD2-CD58 Kd is weak (see references in Ref.
34), relatively strong binding of CD2-CD58 can be achieved by
two-dimensional clustering at cell-cell junctions (32, 36). These CD2
attributes can lower the physiologic TCR activation threshold (32, 37, 38).
Several observations indicate that CD2 participates in a distinct
signaling pathway. First, binding of CD2 and CD58 augments the
interleukin-12 (IL-12) responsiveness of activated T cells (39).
Second, CD2-CD58 interaction plays a role in the reversal of T cell
anergy (40). Third, although the CD2 tail is not responsible for its
adhesion function (32, 41, 42), it is required for optimal CD2
augmentation of antigen-triggered T cell responses (33, 43-46). In
this regard, CD2 has a 117-amino acid cytoplasmic tail that is
relatively conserved among different species (47, 48). Unique pairs of
CD2-specific monoclonal antibodies can transduce mitogenic signals to T
cells and induce T cell proliferation and IL-2 production even in the
absence of engagement of the TCR (33, 49-51). Here we present data
showing that CD2 can inducibly associate with lipid rafts and that raft
integrity is important for CD2 signaling function. We also demonstrate
that CD2-raft association is independent of tyrosine phosphorylation
events as well as the CD2 cytoplasmic tail and TCR linkage to lipid
rafts. Such inducible raft association accounts for many aspects of
CD2-based signal transduction and explains the synergistic activation
of T cells by CD2 and TCR molecules.
Reagents--
Anti-human CD2 mAbs anti-T111
(3T4-8B5) and anti-T112 (1OLD24C1), anti-CD2R mAb
anti-T113 (1mono2A6), and M32B heteroantisera were prepared
and purified as described (41, 49, 52). Anti-T112 Fab fragments were prepared and purified from anti-T112 mAb
using an ImmunoPure Fab preparation kit (Pierce) according to the
manufacturer's protocol. Ascites containing anti-human CD3 Cell Culture--
Human peripheral blood mononuclear cells were
isolated from leukopaks of healthy blood donors by Ficoll
gradient centrifugation using a nylon wool column. The purified
peripheral blood mononuclear cells were cultured in RPMI 1640 medium
containing 10% human AB serum (Nabi, Boca Raton, FL), 1%
penicillin/streptomycin (Life Technologies, Inc.), and 2 mM
glutamine (Life Technologies, Inc.) (complete medium) and used within
0-2 days as resting human T cells. Activated T cells were obtained by
culturing the peripheral blood mononuclear cells as described above
plus a 1:200 dilution of 2Ad2 ascites and 25 ng/ml phorbol 12-myristate
13-acetate. After 48 h of activation, 94% of the cells were
anti-CD2 mAb-reactive as shown by fluorescence-activated cell sorting
analysis (42). Cells were then diluted 1:5, maintained in culture, and
used within 1-3 days. For Ca2+ flux, human T cells were
activated for 48 h, washed once with complete medium, and
cultured in the same medium supplemented with 100 units/ml
recombinant human IL-2 (Amgen, Thousand Oaks, CA), and the cells
were used within 3-6 days.
The human Jurkat cell line J77 was maintained in RPMI 1640 medium and
10% fetal calf serum with 1% penicillin/streptomycin and 2 mM glutamine in 5% CO2 at 37 °C. Chinese
hamster ovary cells (CHOP) without or with CD58 (CHO58) or a
K87A variant (CHO58K87A) were cultured at 37 °C in a 5%
CO2 incubator in Dulbecco's modified Eagle's
medium/nutrient mixture F-12 with 10% fetal calf serum, 1%
penicillin/streptomycin, and 2 mM glutamine and
additionally supplemented with 10 mM HEPES (pH 7.5) and 0.2 mg/ml G418 (Life Technologies, Inc.). The 155.16 mouse T cell hybridoma
cells and human CD2-transfected derivatives were cultured in the same
medium as the J77 cells, but additionally supplemented with 0.5 mg/ml G418.
mAb Cross-linking, T Cell Lysis, and Cell Fractionation--
CD2
cross-linking was achieved by adding either a combination of purified
anti-T112 and anti-T113 mAbs (10 µg/ml) or a
1:100 dilution of ascites. CD3 cross-linking was accomplished by
addition of a 1:200 dilution of 2Ad2 ascites. Cell activation was
performed at 37 °C for 10-15 min in the relevant complete medium.
For pretreatment with various inhibitors, 1 × 107 J77
cells were incubated with 0.1% (v/v) Me2SO for 2 h, 5 µg/ml cytochalasin D for 2 h, 0.5 µM colchicine
for 17 h, 0.5 µM paclitaxel for 17 h, or 20 mM methyl- Lipid Raft Separation--
Sucrose gradient centrifugation was
used to fractionate membrane fractions according to the protocol of
Zhang et al. (53) with some modification. Briefly, 1 × 108 J77 or activated human T cells or 2.5 × 108 resting human T cells were individually harvested,
resuspended in 1 ml of complete medium, and stimulated by cross-linking
as described above. Non-cross-linked samples were treated identically, but without addition of mAbs. After cross-linking, cells were rinsed
once with ice-cold phosphate-buffered saline and then resuspended in 1 ml of ice-cold 1× MES-buffered saline (25 mM MES
and 150 mM NaCl (pH 6.5)) and 1% Triton X-100 supplemented
with 1 mM phenylmethylsulfonyl fluoride, 0.35 trypsin
inhibitor units/ml aprotinin, and 5 µg/ml leupeptin. Following a
30-min incubation on ice, the lysates were homogenized with 10 strokes
in a Dounce homogenizer (Wheaton, Millville, NJ), gently mixed with an
equal volume of ice-cold 80% (w/v) sucrose (Fisher) in 1×
MES-buffered saline, and loaded in the bottom of a centrifuge tube
(Beckman Instruments). The sample was then overlaid with 2 ml of 30%
(w/v) sucrose and 1 ml of 5% (w/v) sucrose, both prepared in 1×
MES-buffered saline. The sucrose gradient samples were spun at 39,000 rpm in a DuPont AH650 rotor at 4 °C for 20-21 h. DIGs were
harvested by collecting 0.4-ml fractions, beginning at the top of the gradient.
Western Blotting--
All cell lysates and gradient fractions
were treated with peptide N-glycosidase F (New England
Biolabs Inc., Beverly, MA) to deglycosylate human CD2 according to the
manufacturer's protocol. Final samples were mixed with 5× SDS loading
buffer and resolved by SDS-PAGE under reducing conditions. Gels were
transferred to polyvinylidene difluoride membranes (Bio-Rad) in
transfer buffer consisting of 25 mM Tris-HCl, 200 mM glycine, and 20% methanol. Membranes were blocked in
5% skim milk in TBS/Tween (25 mM Tris, 150 mM
NaCl, and 0.05% Tween 20) at 37 °C for 30 min. M32B anti-CD2 polyclonal antiserum was diluted 1:1000 in 5% bovine serum albumin and
TBS/Tween and incubated with membranes at 4 °C overnight. The
membranes were then incubated with horseradish
peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad) at
room temperature for 1 h and subsequently developed using a
chemiluminescence reagent kit (PerkinElmer Life Sciences) on MR film
(Eastman Kodak Co.). ECL-developed blots were scanned with a Microtek
ScanMaker 5 and processed with Photoshop software. The intensity of
anti-T112 and anti-T113 mAb cross-linking in
the presence of the control (Me2SO or no other addition)
was arbitrarily set to 100% using ImageQuant Version 1.1 (Molecular
Dynamics, Inc.).
For phosphotyrosine blotting, activated human T cells were left
untreated or were pretreated with Generation of T Cell Transfectants Expressing Mutant CD2
Molecules--
Wild-type CD2 transfectants as well as mutant CD2
molecules (including the cytoplasmic deletion CD2 Ca2+ Flux--
1 × 106 activated
human T cells (see above) were loaded with 2 µg/ml (~2
nM) Indo-1 acetoxymethyl ester (Molecular Probes, Inc.,
Eugene, OR) in 1 ml of complete medium at 37 °C for 30 min. Following three washes with RPMI 1640 medium, cells were resuspended in
RPMI 1640 medium and prewarmed for 5-10 min at 37 °C before analysis. Ca2+ flux was measured on a FACS Vantage (Becton
Dickinson, San Jose, CA). Samples were run for 30 s to obtain a
base line, and then a combination of anti-T112 and
anti-T113 ascites (1:100 dilution of each) was added. The
ratio 405/525 nm was recorded for 8 min. 2 µg of 4-bromo
A23187 (calcium ionophore) (Molecular Probes, Inc.) was added
subsequently to ensure the successful loading of Indo-1. For Cross-linking-mediated Translocation of Human CD2 to the
Detergent-insoluble Fraction--
Previous studies from our laboratory
have indicated that CD2 cross-linking by mitogenic pairs of mAbs
(anti-T112 and anti-T113) results in loss of a
fraction of CD2 molecules from the 1% Triton X-100 detergent-soluble
component of T cell lysates.3
Hence, CD2 must become associated with detergent-insoluble cellular components such as nuclei, the cytoskeleton, and/or specialized domains
of the plasma membrane. To investigate the basis of this phenomenon, we
pretreated J77 cells prior to CD2 stimulation with chemicals known to
modulate these components. These reagents included cytochalasin D,
which blocks the formation of actin filaments; colchicine, which
inhibits microtubule formation; paclitaxel (Taxol), which stabilizes
microtubule structures; and
As shown in Fig. 1, without CD2
cross-linking, CD2 partitioned entirely in the detergent-soluble
lysate; and hence, none was detected in the detergent-insoluble
fraction (lane 7). By contrast, following CD2
cross-linking, some CD2 was found in the insoluble fraction (lane
1). Interestingly, stimulation of J77 cells with mitogenic
anti-CD3 Inducible Association of CD2 with Lipid Rafts--
Since plasma
membrane lipid rafts have distinct lipid and protein contents, rafts
are easily separated by sucrose gradient centrifugation (10). We took
advantage of this methodology to directly and independently examine if
CD2 can be recruited to lipid rafts. To this end, activated human
peripheral blood T cells either were stimulated by the combination of
anti-T112 plus anti-T113 mAbs or were left
untreated at 37 °C for 10 min. Total cell lysates were generated by
extracting cells in a mild nonionic detergent (1% Triton X-100) at
4 °C. Sucrose gradient fractionation and subsequent Western blotting
analysis were performed as described under "Experimental Procedures."
Consistent with previous reports, DIGs are found in sucrose gradient
fractions 2-4 (22, 53), where the palmitoylated LAT molecule is
predominantly located (53). Other proteins including A Mitogenic Pair of Anti-CD2 mAbs Is Necessary for CD2-Lipid Raft
Association--
The above results indicate that CD2 association with
lipid rafts is stimulated by the combination of anti-T112
plus anti-T113 mAbs. In this regard, it was previously
shown that there are at least three distinct epitopes on the human CD2
molecule (49): the T111 epitope localized to the
CD58-binding face; the T112 epitope adjacent to the
CD58-binding site; and the T113 epitope, termed CD2R, which
is mainly restricted to activated human T cells, unlike the
T111 and T112 epitopes, and involves the linker
region between domains 1 and 2 of CD2 (41). Jurkat cells were treated with different combinations of antibodies, and then rafts were fractionated by sucrose gradient centrifugation. The various DIG fractions were analyzed by Western blotting as shown in Fig.
3A. As expected, anti-T112 plus
anti-T113 mAb cross-linking recruited CD2 to lipid rafts
(lane 1). Earlier studies demonstrated that bivalent
anti-T112 mAb, but not monovalent Fab, induced
expression of CD2R (41, 49) and, furthermore, that
anti-T112 Fab plus anti-T113 IgG was
non-mitogenic (41). As shown in Fig. 3A (lane 2),
when we tested anti-T112 Fab in combination with
anti-T113 mAb, no CD2-raft association was induced,
consistent with the inability of this combination to stimulate
proliferation. Given that anti-T112 mAb alone was not
capable of recruiting CD2 to DIG (lane 3), we can also
exclude Fc receptor binding as the basis of CD2 translocation. These
results show that neither anti-T112 nor
anti-T113 mAb alone is sufficient to translocate CD2 to
lipid rafts.
As the binding of anti-T112 and anti-T113 mAbs
is essentially orthogonal to one another, the combination of the
two mAbs presumably supports extensive lattice formation (41). To
examine whether other forms of significant CD2 cross-linking could
recruit CD2, we bound biotinylated anti-T112 mAb to J77
cells and then cross-linked the CD2 by subsequent addition of avidin.
Fig. 3A (lane 4) shows that there was no CD2
translocation to the raft fraction after such treatment, consistent
with the notion that specific cross-linking of CD2 by the mitogenic
combination of anti-T112 plus anti-T113 mAbs is
required. The combination of anti-T111 plus
anti-T112 mAbs failed to recruit CD2 to lipid rafts as
well, consistent with this view (lane 5).
Additional data supporting the selective CD2 translocation-inducing
action of the combination of anti-T112 plus
anti-T113 mAbs derive from analysis of mouse T cell
hybridomas transfected with variant CD2 molecules involving mutation at
the T112 or T113 epitopes (41). As shown in
Fig. 3B, for example, CD2 molecules harboring the triple
mutant K61E/F63L/T67D, which disrupts anti-T112 binding
while retaining anti-T111 and anti-T113 binding
activity, failed to be recruited to lipid rafts upon
anti-T112 plus anti-T113 mAb addition. Other
CD2 mutants that disrupt anti-T113 binding (including
K167W, K167A, K170A, W10F, E8A, and H140A) also failed to move into
DIGs with the same mAb cross-linking pair. On the other hand, the CD2
double mutant N117Q/N126Q, lacking N-linked adducts through
modification of the two glycan addition sites within the
membrane-proximal domain 2 of CD2 (without perturbing T111-, T112-, or T113-binding
sites), and the CD2 variant E131A, both of which retain
anti-T112- and anti-T113-binding sites,
associated with rafts upon addition of the anti-T112 plus
anti-T113 mAb combination. The above results illustrate
that both anti-T112 and anti-T113 mAbs are
necessary and sufficient to recruit wild-type CD2 molecules or CD2
variants bearing T112 and T113 epitopes to
lipid rafts. The other combinations of these anti-CD2 mAbs tested with
or without additional CD2 cross-linking are ineffective.
Lipid Rafts Play a Key Role in CD2 Signaling--
The above
results demonstrate that translocation of CD2 into lipid rafts requires
a specific pair of anti-CD2 antibodies known to transduce mitogenic
signals. Given that lipid rafts are rich in T cell signaling molecules,
these findings raise the question as to whether lipid rafts contain
critical signaling components for the CD2 transduction pathway. To this
end, we examined whether CD2 signaling is altered by perturbation of
raft structures. We utilized
By reblotting the above membrane with antibodies specific for
individual signaling molecules, we were able to examine the status of
each after
Since phospholipase C Independent Recruitment of CD2 and CD3 Molecules to Lipid
Rafts--
TCR cross-linking by mAbs or engagement with the pMHC was
previously reported to recruit TCRs to lipid rafts (17, 22). Co-stimulatory molecules such as CD28 (29), CD5, and CD9 exert their
co-stimulatory functions, at least in part, by contributing to enhanced
association between the TCR and lipid rafts (28). To determine whether
CD2 mediates its co-stimulatory function by a similar mechanism, we
stimulated activated human T cells with either anti-CD2 or anti-CD3 mAb
cross-linking, separated raft fractions by sucrose gradient
centrifugation, and analyzed them for inducible association with CD2
and/or CD3 by Western blotting. Consistent with previous reports, we
failed to observe CD3 redistribution to the lipid raft fraction in the
presence of 1% Triton X-100 (data not shown). This lack of association is probably due to the weak interaction between the TCR components and
lipid rafts (8, 22) since, by lowering the concentration of Triton
X-100 to 0.2%, we were able to observe that significant amounts of
CD3
Since TCR recruitment to rafts requires tyrosine phosphorylation events
(22), it was of interest to determine if, by analogy, similar
phosphorylation was required for CD2-DIG association. In this regard,
the CD2 tail can bind to Src family kinases, including Fyn (63) and Lck
(64), which are known to be localized in rafts, as well as
phosphatidylinositol 3-kinase (65), which is recruited to rafts
upon T cell activation (17). As shown in Fig. 5B, PP2
treatment of cells could not block CD2 recruitment to lipid rafts, even
though it virtually eliminated CD2-triggered protein tyrosine
phosphorylation (Fig. 4A, lane 5). Thus, tyrosine phosphorylation and CD2 translocation to DIGs are separable events. Similar phosphorylation-independent CD2 relocation to lipid rafts was
observed in freshly isolated resting human T cells and in primary
murine T cells from human CD2 transgenic mice with or without an
accompanying fyn gene knockout (data not shown).
The CD2 Extracellular Domain Is Required for Lipid Raft
Recruitment--
To next determine the domain(s) of CD2 responsible
for raft association, we tested a series of mutant and truncated CD2
molecules, including CD2 CD2 Translocation to DIG Is Independent of the Membrane-proximal
Domain Glycans--
In non-lymphoid cells, it has been reported that
N-glycans on membrane proteins can mediate raft association
through binding to resident lectin-like molecules within lipid rafts
(66, 67). To assess whether this might be the case for CD2 in lymphoid
cells, we used three mutant CD2 molecules in which one or the other
complex N-linked CD2 glycans (N117Q and N126Q) or both
(N117Q/N126Q) were eliminated (41). Under these circumstances,
recruitment of mutant CD2 molecules into lipid rafts was still observed
after antibody cross-linking (Fig. 6D), implying that the
membrane-proximal glycans are not required to induce CD2 association
with DIG.
CD58 Ligation Is Sufficient to Recruit CD2 to Lipid
Rafts--
Although the above experiments identified a role for a
specific pair of anti-CD2 mAbs in inducing CD2 translocation to DIG, it
remained to be determined whether a similar translocation might be
induced by the physiologic human CD2 ligand, CD58. To address this
question, we utilized CHO cells as a simplified version of antigen-presenting cells. A CHO cell transfected with the expression vector alone (CHOP) was used as a negative control, whereas CHO cells
transfected with the glycosylphosphatidylinositol-linked form of human
CD58 (CHO58) were used as the ligand-positive control. The CHO cells
were mixed with J77 cells at a 1:5 cell/cell ratio, spun down, and
resuspended in a small volume of warm medium to facilitate cell-cell
conjugate formation. The cell-cell conjugates were formed at 37 °C
for 5 min, and then cells were quickly collected by spinning, followed
by raft isolation as described above. As shown in Fig.
7A, there was a 2.3-fold
increase in the amount of CD2 localized to rafts upon CD58 ligation in
comparison with the negative control CHOP cells. These findings
indicate that CD2 binding to CD58 is able to induce translocation of
CD2 into lipid rafts. That comparable amounts of raft fractions were
loaded from T cells contacting CHOP and CHO58 APCs was verified by
reblotting the raft fraction with anti-LAT antibody (Fig.
7A).
We further confirmed the above result using a CHO cell transfectant
bearing a single point mutation (K87A) of CD58 and defective in CD2
binding (68). To demonstrate that these observations were not
restricted to J77 cells, we employed activated human T cells. As shown
in Fig. 7B, compared with CHO58K87A cells, there was a
2.5-fold increase in CD2 recruitment to rafts by T cells when
conjugated with CHO58 cells. By comparing the ratio of CD2 in
the raft fraction versus CD2 in the soluble fraction, we can conclude that the extent of raft recruitment induced by CD58 is about
an order of magnitude weaker compared with antibody-induced cross-linking (compare Figs. 2 and 7). This result is not surprising given that the affinity of the CD2-CD58 interaction is 100-fold less
than that of the CD2-mAb interaction.
Inducible Nature of CD2-Raft Association--
In this study, we
show that human CD2 is not constitutively associated with membrane
rafts, consistent with earlier reports (69). More importantly, we
demonstrate, for the first time, that CD2 is inducibly recruited to
lipid rafts after CD2 cross-linking by specific anti-CD2 mAbs or
CD58-bearing APCs. Most membrane raft-associated proteins are
constitutive residents because of glycosylphosphatidylinositol
modification or through palmitoylation (7, 10). Included among the
latter are G proteins (16, 17), the Src family protein-tyrosine kinases
(p56lck and p59fyn) (14, 15), and CD4 and LAT (53).
Each of these proteins is palmitoylated at membrane-proximal cysteine
residue(s) (reviewed in Ref. 20). By contrast, the human CD2 protein
lacks any cysteine residues in its cytoplasmic domain and hence cannot
be palmitoylated. In this regard, it is particularly significant that
there are two membrane-proximal cysteines in the murine and rat CD2
tails that are absent from horse and human homologs (Fig.
6A) (47, 48). This species difference is noteworthy since it
was previously reported that substantial amounts of mouse CD2 are
raft-associated in resting murine lymphocytes (28). The evolution of
CD2 membrane partitioning to a raft-inducible rather than constitutive
association mechanism may offer a partial explanation for any
functional distinctions in the role of CD2 in man and mouse,
respectively. In addition, mouse CD48 is a weaker ligand for mouse CD2
than human CD58 is for human CD2 (by at least an order of magnitude)
and has a more restricted distribution (35, 70). Perhaps the
constitutive membrane partitioning of mouse CD2 compensates for this
lower affinity ligand interaction.
Distinctions between CD2- and TCR-inducible Membrane Raft
Association--
Inducible association with lipid rafts upon ligand
binding or antibody cross-linking has been demonstrated for several
other surface molecules. The first studies involved Fc
The CD2-raft association observed here is different from the above
immunoreceptor complexes based on its stability under various extraction conditions. In particular, the CD2-raft interaction is more
robust than the TCR, B cell receptor, or Fc
The recruitment of CD2 to lipid rafts is independent of the cytoplasmic
tail segments (amino acids 8-117) known to be involved in CD2
signaling function. In fact, this very segment has been shown to bind
to important signaling and/or adaptor molecules, including the Src
family kinases p59fyn and p56lck (63, 64);
phosphatidylinositol 3-kinase (65); and the CD2 tail-binding molecules
CD2BP1 (42), CD2BP2 (72), and CD2AP (73). Such a result is distinct
from the TCR behavior given that 1) recruitment of the TCR to lipid
rafts is Src family kinase-dependent; 2) TCR-induced raft
redistribution can be inhibited by Src-type tyrosine kinase inhibitors
such as PP1 (22); and 3) these events are also blocked by wortmannin,
an inhibitor of phosphatidylinositol 3-kinase. Triggering through the
TCR by anti-CD3
These data indicate that TCR interactions with rafts are downstream of
prerequisite tyrosine phosphorylation events. In contrast, pretreatment
of activated human T cells with tyrosine kinase inhibitors does not
affect CD2-raft redistribution (Fig. 5B). Moreover, all known CD2 tail associations with signaling pathways (43, 44) are
eliminated by the CD2
Inducibility of the CD2R epitope in the CD2 ectodomain upon specific
anti-CD2 mAb cross-linking as well as by the CD58 ligand (41, 49), both
stimulators of CD2 translocation to lipid rafts, suggests that
ectodomain conformational change upon ligation is responsible, at least
in part, for the CD2-DIG translocation. We cannot exclude a role for
the membrane-proximal seven CD2 cytoplasmic residues since deletion of
these residues reduces surface CD2 expression to a level too low to
accurately measure translocation quantitatively. Likewise, although the
sialic acid-containing membrane-proximal domain glycans at
Asn117 and Asn126 were excluded by mutagenesis
as important for raft translocation, the high mannose glycan in domain
1 was not eliminated given that removal of the latter would disrupt the
native fold of CD2 domain 1 (41). Interaction of a resident DIG
lectin-like protein with this domain 1 glycan might theoretically offer
a basis for translocation (66, 67), but as this simple glycan is
exposed on resting T cells, such a docking could not explain the
inducible CD2 translocation behavior observed.
Lipid Raft Association Versus Mitogenicity--
The mAb
requirement for CD2 translocation to lipid rafts identically
parallels the requirement for antibody-induced mitogenicity. Hence, to
induce CD2 translocation or T cell proliferation, binding of the
anti-T112 plus anti-T113 mAb pair is required.
This observation suggests that the two processes are linked,
particularly given the requirement for intact lipid rafts to mediate
CD2-triggered activation function (Fig. 4) and the known raft
association of substrate and enzyme components involved in
Ca2+ flux, IL-2 gene activation, and T cell proliferation.
On the other hand, we failed to observe mitogenicity upon exposure of T
cells to CD58-expressing antigen-presenting cells, although such
cell-cell conjugates trigger CD2 translocation to DIG (Fig. 7). Such a
disparity between anti-CD2 mAb- and CD58-triggered translocation of CD2
may be a consequence of differences both in the duration of the
interaction and in the extent of raft association. Rapid
kon and koff rates of
CD58 (35) compared with anti-CD2 mAbs imply that the CD2-ligated state
is short-lived in the case of the natural ligand. In fact, the dynamic
binding of the CD2-CD58 co-receptor pair is important; this
characteristic facilitates adhesion (micromolar Kd),
but does not impede diffusion of TCRs into the abutting T cell-APC
membranes in a given cell-cell conjugate. In turn, and possibly as a
consequence of the binding kinetics, the amount of CD2 associated with
lipid rafts following CD58 ligation of CD2 represents only 10% of the
CD2 molecules induced to translocate to DIG by anti-CD2 mAbs (compare
Figs. 2 and 7). The CD2 association with rafts is transient, being
substantially reduced by 30 min following ligation with CHO58 cells
(data not shown). In this regard, prior studies of T cell proliferation have shown that a potent stimulating signal needs to be sustained for
hours to maintain gene transcription and to promote T cell cycle
progression, with the cumulative amount of signal received determining
the fate of T cell activation (74). Such transient interaction between
CD2 and rafts upon CD58 ligation noted herein may provide initial
signals for activation, but not, by itself, achieve the threshold
required to trigger T cell proliferation. Nevertheless, CD58 ligation
is known to activate itk (75), to stimulate IL-12
responsiveness (39), and to reverse anergy (40).
The Specific Nature of CD2 Cross-linking Stimuli Required for DIG
Translocation and Mitogenicity--
As described in Fig. 3, the
anti-CD2 mAb requirement for CD2 translocation to DIG and mitogenicity
is highly specific. Cross-linking of CD2 molecules per se is
not sufficient either for translocation or mitogenicity. Hence,
anti-T112 or anti-T113 mAb alone or with secondary anti-mouse Ig cross-linking, cross-linking of a biotinylated anti-T112 mAb with avidin, and cross-linking by rabbit
anti-human CD2 heteroantisera were all insufficient to induce raft
translocation or T cell proliferation despite extensive capping of CD2
observed microscopically in the latter two cases (Fig. 3, Ref. 41, and data not shown). These findings suggest that cross-linking of CD2 must
be achieved to generate a molecular lattice with CD2 molecules in a
specified conformation and/or distance from other CD2 molecules (41).
The importance of one of the two mAbs in a mitogenic anti-CD2 mAb pair
mapping to Phe54 of CD2 domain 1 is consistent with this
notion (76). Such a precedent for defined cross-linking has been
observed in the erythropoietin (EPO) receptor system (77, 78). The EPO
receptor is a dimer even in the absence of its ligand EPO (77). Upon
EPO binding, however, the receptor undergoes a conformational change
resulting in rearrangement of the signaling tail such that JAK2 can be
autophosphorylated and transduce the EPO signal (78). The
EPO-mimetic peptides that function as antagonists permit the
receptor to dimerize, but in a different orientation from that of EPO.
This orientational difference precludes productive signaling through
the receptor; and hence, EPO receptor-based signaling is inhibited.
Assuming that there is a specific set of CD2 conformational and/or
distance requirements for CD2 lattice formation resulting in signaling upon CD2 cross-linking, then the combination of anti-T112
plus anti-T113 mAbs, but not random forms of
antibody-mediated cross-linking, will result in raft association or T
cell mitogenicity. Given that there are other combinations of anti-CD2R
plus anti-CD2 mAbs that can trigger a mitogenic response (antibodies
9.1 and 9.6, respectively) (51) as well as CD2 translocation to
DIG (data not shown), these reagents may induce a cross-linking lattice equivalent to that stimulated by anti-T112 plus
anti-T113 mAbs and distinct from other antibody pairs.
CD2-based Membrane Reorganization and Raft Coupling Prior to pMHC
Complex Recognition: A Mechanism to Facilitate TCR Signaling--
The
movement of TCRs to DIG requires tyrosine phosphorylation by kinases
that are resident within the lipid rafts (22). In turn, the integrity
of the lipid rafts is essential for TCR signal transduction (17, 22).
Thus, the question remains as to how a TCR initially becomes linked to
DIG. That CD2 molecules redistribute to the membrane cell-cell
conjugation surface (32, 41) and, upon CD58 ligation, couple to lipid
rafts in the absence of phosphorylation events offers one explanation.
CD2-based adhesion is pMHC-independent and occurs prior to and without
any requirement for antigen-triggered immune responses (6, 32, 33). The CD2-enriched, DIG-coupled cell surface membrane patch is ideally designed to facilitate TCR contact with the pMHC at the intercellular contact zone by promoting dynamic adhesion between abutting membranes with a spacing suitable for TCR-pMHC interaction (Ref. 34 and references therein). Recent microscopic analysis of murine T cell recognition processes has shown that cell surface molecules are redistributed into supramolecular activation clusters (79) with a
central accumulation of TCR-pMHC complexes surrounded by CD2-CD48 complexes and a more peripheral ring of LFA-1/ICAM-1, also referred to
as an "immunologic synapse" (80, 81). Given the aforementioned species differences, it remains to be determined whether a similar supramolecular activation cluster morphology is observed during human T
cell-APC interaction. Note that this supramolecular organization occurs
as the consequence of TCR triggering rather than being the basis of
such activation (79, 80).
Because of the inducible human CD2 coupling to lipid rafts,
p56lck and p59fyn kinases are pre-configured to
participate in the initial TCR activation process. These enzymes as
well as their adaptors (endogenous or associated) may facilitate
subsequent recruitment of TCRs to DIGs in the T cell-APC contact zone,
hence favoring further activation events. The ability of CD2
cross-linking to synergistically facilitate triggering through the TCR
(51) may be reflective of this "shared" raft coupling phenomenon
involving distinct translocation mechanisms. Further studies in this
regard are clearly warranted.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor I)
(26, 27) are recruited to rafts and that raft integrity is required for
effective signal transduction (reviewed in Ref. 9). A number of
co-stimulatory molecules on the T cell surface can apparently
up-regulate TCR signals by enhancing association of the TCR and lipid
rafts. Representative molecules of the latter type include CD5 and CD9
(28) and CD28 (29).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mAb 2Ad2
(IgM) were produced in our laboratory. mAb 4G10 (anti-phosphotyrosine)
was kindly provided by Dr. Tom Roberts (Dana-Farber Cancer Institute). Antisera against p59fyn were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-phospholipase C
1 mAb and
antisera against LAT and Vav were obtained from Upstate Biotechnology,
Inc. (Lake Placid, NY). Cytochalasin D, colchicine, paclitaxel,
methyl-
-cyclodextrin, n-octyl
-D-glucopyranoside, and Triton X-100 were purchased from
Sigma. PP2
(4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4d]pyrimidine) was from Calbiochem, and Nonidet P-40 was the product of U. S. Biochemical Corp. (Cleveland, OH). All other chemicals were purchased from either Sigma or Fisher.
-cyclodextrin (
-MCD) for 30 min at 37 °C
in complete J77 medium. Cells were spun down at 1000 × g and resuspended in medium with or without antibody
stimulation. After 15 min at 37 °C, cells were washed with ice-cold
phosphate-buffered saline and lysed in 0.5 ml of lysate buffer
containing 1× TBS and 1% Nonidet P-40 supplemented with 1 mM phenylmethylsulfonyl fluoride, 0.35 trypsin inhibitor
units/ml aprotinin (Sigma), and 5 µg/ml leupeptin (Roche Molecular
Biochemicals). Following incubation on ice for 30 min, samples were
spun down at 12,000 × g for 10 min. Cell pellets were
resuspended in lysate buffer containing 0.1% SDS and 0.5%
deoxycholate, followed by brief sonication. Supernatants were recovered
as the insoluble fraction of the cell lysate and quantified with a
Micro BCA kit (Pierce). Equal amounts of total protein were loaded per
lane for Western blot detection of CD2.
-MCD or PP2, followed by cross-linking of CD2 or CD3. Whole cell lysates were generated in lysis
buffer containing 1× TBS and 60 mM n-octyl
-D-glucopyranoside supplemented with protease
inhibitors. Equal amounts of lysate proteins were loaded on 4-12%
gradient SDS-polyacrylamide gels (Bio-Rad) and then transferred to
polyvinylidene difluoride membranes as described above. The membranes
were blocked in 5% bovine serum albumin and TBS/Tween at 37 °C for
30 min and subsequently incubated with antibody 4G10 at 4 °C
overnight. 4G10 signals were detected with horseradish
peroxidase-conjugated goat anti-mouse IgG2b, followed by ECL. The
membrane was then stripped with 1× stripping buffer (Chemicon
International, Inc., Temecula, CA) and reblotted with antibodies
against Fyn, LAT, Vav, and phospholipase C
1.
25; the single
mutation variants K167W, K167A, K170A, W10F, E131A, E8A, N117Q, N126Q, and H140A; the double mutant N117Q/N126Q; and the triple mutant K61E/F63L/T67D) were generated as described (41). TfR25 and TfRTM are two variants in which the CD2 transmembrane
domain was swapped with that of the transferrin receptor (TfR). These
mutants were produced using a one-step reverse transcription-polymerase chain reaction (Life Technologies, Inc.) to clone the cDNA encoding the transmembrane domain of the TfR, which was subsequently fused by
additional polymerase chain reaction cloning with the extracellular domain of CD2 with (TfR25) or without (TfRTM) a
cDNA sequence encoding 25 amino acids of the CD2 cytoplasmic
tail.2 Subsequently, the
desired DNA sequence was verified, and the product was cloned into the
expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA). The final
expression vectors were linearized by SalI digestion and
transfected into the 155.16 mouse T cell hybridoma cells by
electroporation at 200 V and 1180 microfarads (Life Technologies, Inc.). Following neomycin selection (1.5 mg/ml), stable transfectant cell lines expressing CD2 mutants at levels comparable to wild-type CD2
were established by sorting four to five times on a MoFlo (Cytomation,
Fort Collins, CO).
-MCD
treatment, different concentrations of
-MCD were added to the
loading buffer concurrently with Indo-1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MCD, known to specifically extract
membrane cholesterol and therefore disrupt plasma membrane lipid rafts
(54). Cell viability was not affected by any of these treatments as
indicated by trypan blue exclusion (data not shown); following
treatment, the cells were then stimulated with anti-T112
plus anti-T113 mAbs; cell pellets were generated; and proteins were deglycosylated with peptide N-glycosidase F
under denaturing conditions as described under "Experimental
Procedures." Deglycosylation facilitates quantitative analysis of the
CD2 protein amount by collapsing the various species bearing distinct
glycan adducts into a single band. Equivalent amounts of protein were resolved by SDS-PAGE, and CD2 was quantitated by Western blotting.
mAb 2Ad2 (IgM) did not shift the CD2 into the insoluble
cell pellet (lane 6), indicating that CD2 association with
the detergent-insoluble fraction is independent of TCR cross-linking and the attendant cell activation induced via anti-CD3 mAb.
Perturbation of the cytoskeleton by the inhibitors cytochalasin D and
colchicine or, conversely, by the stabilizer paclitaxel did not affect
the partitioning of CD2 into the insoluble cell pellet (lanes
2-4). On the other hand,
-MCD prevented >50% of the CD2 from
associating with the insoluble fraction after CD2 cross-linking
(lane 5). This finding suggests that some CD2 molecules
become linked to lipid rafts upon anti-CD2 mAb cross-linking since the
above cytoskeletal actin and microtubule modifiers were without effect.
Although we interpret the influence of
-MCD on CD2 localization to
reflect its disrupting effect on lipid rafts, the cholesterol-depleting activity of the compound might modify membranes elsewhere in the cell,
making the conclusion tentative.
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Fig. 1.
Partitioning of CD2 in the insoluble fraction
of T cell lysates upon cross-linking. J77 cells were pretreated
with the indicated reagents (Me2SO solvent control
(DMSO; 0.1% (v/v), 2 h), cytochalasin D
(cyto-D; 5 µg/ml, 2 h), colchicine (COL;
0.5 µM, 17 h), paclitaxel (TAX; 0.5 µM, 17 h), and -MCD (20 mM, 30 min))
or were left untreated (
) at 37 °C. Surface molecules were then
cross-linked with either a combination of anti-T112 plus
anti-T113 mAbs (10 µg/ml) (lanes
1-5), mAb 2Ad2 (1:100; anti-CD3
ascites) (lane 6),
or were untreated (lane 7). Subsequently, cells were lysed
in 1% Triton X-100 and centrifuged, and cell pellets were resuspended
in 0.1% SDS and 0.5% deoxycholate buffer and solubilized by brief
sonication. Equal amounts of proteins from each condition were treated
with peptide N-glycosidase F (PNGase F) and then
resolved by SDS-PAGE. CD2 was visualized by M32B polyclonal antibodies,
followed by horseradish peroxidase-labeled goat anti-rabbit antibody
and ECL.
-tubulin are
not associated with lipid rafts (22, 24), but rather are located in the
soluble cellular fractions. As shown in Fig.
2, following CD2 cross-linking,
substantial amounts of CD2 (~32-fold increase) were recruited to the
lipid raft fraction, whereas in the absence of CD2 cross-linking,
little if any CD2 was localized to the rafts. This result indicates
that CD2 association with lipid rafts is inducible. Note that although
the majority of LAT is found in DIG as reported (53), the recruited CD2
component represents only 5-10% of total cellular CD2. Although these
data were generated using activated T cells, similar results were
obtained using resting human T cells, the human J77 tumor cell line,
human CD2 transgenic primary mouse T cells (55), and human CD2
cDNA-transfected 155.16 T cell hybridoma cells (Figs.
3 and 6, Ref. 41, and data not
shown).
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Fig. 2.
CD2 inducibly associates with lipid
rafts. 8 × 107 activated human T cells were
either cross-linked with a combination of anti-T112 + anti-T113 mAbs (1:100; ascites) (A) or left
untreated (B) at 37 °C for 10 min. Total cell lysates
were fractionated on sucrose gradients, and then 0.4 ml of each
fraction was collected, treated with peptide N-glycosidase
F, and resolved by SDS-PAGE. CD2 was Western-blotted (WB)
with M32B polyclonal antibody and visualized as described in the legend
to Fig. 1. Membranes were subsequently stripped and reblotted
(RB) with anti-LAT polyclonal antibodies or anti- -tubulin
mAb to verify positions of raft and soluble fractions,
respectively.
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Fig. 3.
Specific anti-CD2 antibody requirements for
induction of CD2-lipid raft association. A, 8 × 107 J77 cells were treated with the indicated combinations
of anti-CD2 mAbs (10 µg/ml) at 37 °C for 10 min. Cell lysates were
then fractionated on sucrose gradients, and the raft component was
collected and subjected to Western blot (WB) analysis with
anti-CD2 heteroantisera. The membranes were stripped and reblotted
(RB) with anti-LAT antisera to demonstrate equivalent
loading of raft fractions. Band intensity <20% of the
anti-T112 + anti-T113 mAb-induced sample is
considered as background in the mAb-triggered responses of wild-type
CD2 molecules. B, 1 × 108 155.16 mouse T
hybridoma cells transfected with human CD2 bearing the indicated
mutations were treated (+) with anti-T112 + anti-T113 mAbs (10 µg/ml) or were untreated ( ) and were
processed as described for A. Note that only the raft
fractions are shown. Bio, biotinylated.
-MCD, a cyclic oligosaccharide
consisting of seven glucopyranose units that extracts cholesterol from
the plasma membrane to disrupt the raft structures (54). Activated
human T cells were treated with
-MCD or left untreated, and then
cells were stimulated via the CD2 or CD3 pathways with anti-CD2 or
anti-CD3 mAbs, respectively. Fig.
4A shows that upon CD2
cross-linking, multiple proteins were tyrosine-phosphorylated as
identified by anti-phosphotyrosine mAb 4G10 in Western blotting
(compare lanes 1 and 2). Following anti-CD3
mAb-mediated cross-linking, certain proteins (including CD3
and LAT)
were more heavily phosphorylated than upon CD2 stimulation (compare
lanes 2 and 3). Importantly, pretreatment of T
cells with
-MCD reduced tyrosine phosphorylation (compare lane
4 and lanes 1 and 2). The strong Src family
tyrosine kinase inhibitor PP2 was used as an additional control
(lane 5) and virtually abrogated tyrosine phosphorylation
triggered by anti-CD2 mAb, reducing phosphorylation to below the basal
level of unstimulated cells. This result is consistent with previous
reports showing that PP2 is able to block early T cell activation
events (56).
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Fig. 4.
Disruption of lipid rafts impairs
CD2-mediated signal transduction. A, activated human T
cells were cross-linked with anti-T112 + anti-T113 mAbs (CD2-XL) or anti-CD3 mAb 2Ad2
(CD3-XL) either treated with or without
-MCD (15 mM, 37 °C, 30 min) or PP2 (10 µM,
37 °C, 10 min). Cells were lysed using n-octyl
-D-glucopyranoside. Equal amounts of lysate proteins (12 µg) were loaded on 4-20% gradient SDS-polyacrylamide gel and then
subjected to Western blot (WB) analysis with
anti-phosphotyrosine mAb 4G10. The same membrane was then stripped and
reblotted (RB) with the indicated specific antibodies.
Numbers under each lane represent the intensity of
phosphoprotein (P) relative to the unstimulated control (set
at 1.0). PLC-
1, phospholipase C
1.
B, human T cells were assayed for Ca2+ flux as
described under "Experimental Procedures." Representative
histograms of the ratio 405/525 nm are shown. C, shown
is the dose-dependent inhibition of
-MCD on
Ca2+ flux. The black arrow indicates the time of
addition of anti-T112 + anti-T113 mAbs (30-s
point), and the white arrow indicates the time of addition
of the calcium ionophore (A23187; 8-min point). The ordinate
(
MFI) represents the change at each point
after background subtraction.
-MCD treatment. The Src family tyrosine kinase
p59fyn was shown to be critical in the CD2 signaling, so in
fyn
/
mice, murine T cells
demonstrated impaired CD2 signaling in terms of Ca2+ flux,
T cell proliferation, and tyrosine phosphorylation (55). The current
results show that
-MCD treatment decreased phosphorylation of
p59fyn 2-fold (Fig. 4A, lane 2 versus
lane 4), reflecting reduced activity of the p59fyn
kinase for CD2 stimulation. The LAT scaffold protein, a substrate of
the ZAP70 family of kinases, plays a central role in T cell activation (57, 58). Although there is a severalfold increase in
phospho-LAT upon CD2 stimulation,
-MCD treatment diminished this
phosphorylation by 50%. Another intracellular signal molecule affected
by
-MCD treatment is Vav. Vav is known to be involved in both T cell
development and antigen receptor-mediated T cell activation due to its
ability to trigger activation of ERK and NFAT as well as NF-
B (59).
Vav has also been shown to be essential for actin cytoskeletal
reorganization and TCR capping (60, 61). As shown in Fig.
4A, Vav was phosphorylated upon CD2 or CD3 stimulation, whereas virtually no phospho-Vav was detected following
-MCD pretreatment of anti-CD2 mAb-triggered T cells. This result is consistent with our recent report that the Vav signaling pathway is
important for CD2-triggered T cell activation (55). The tyrosine phosphorylation of phospholipase C
1 was also severely impaired after
-MCD treatment (Fig. 4A), suggesting that the enzymatic activity of phospholipase C
1 is likewise decreased (62).
1 plays a critical role in calcium
mobilization, CD2-triggered Ca2+ flux could be affected by
disruption of CD2 association with lipid rafts. To test this
possibility, we loaded activated human T cells with Indo-1 and, in some
cases, pretreated them with different concentrations of
-MCD. Cells
were then analyzed by fluorescence-activated cell sorting and subjected
to CD2 stimulation by anti-T112 plus anti-T113
mAb cross-linking. The ratio 405/525 nm was recorded as an
indication of Ca2+ flux. Fig. 4B shows two
typical histograms of CD2 stimulation of T cells with or without
-MCD (2 mM) treatment. After
-MCD treatment, the
Ca2+ flux was decreased relative to the untreated T cell
control. Subsequent addition of Ca2+ ionophore indicated
that both
-MCD-treated and untreated cells were loaded with Indo-1
to a similar extent. This decrease in CD2-triggered Ca2+
flux was
-MCD dose-dependent, as shown in Fig.
4C. A decrease in calcium flux was evident at a
-MCD
concentration as low as 0.5 mM and was almost completely
inhibited at 8 mM. Thus, two early T cell activation
events, tyrosine phosphorylation and a rise in intracellular-free
calcium, were both impaired upon CD2 stimulation following perturbation
of lipid rafts by
-MCD. These results indicate that the integrity of
the lipid raft structure is important for CD2 signal transduction.
Collectively, these data show that, in addition to TCR signaling, CD2
signaling requires a functional lipid raft membrane microdomain.
were recruited to DIGs (Fig.
5A). Upon CD2 cross-linking, however, there was no concurrent translocation of CD3 to the lipid raft
fraction, although CD2 could be readily found associated with DIG.
Conversely, upon anti-CD3
mAb cross-linking, there was no increase
in CD2 in the DIG fraction. The equivalent loading of raft fractions
was evident from the reanalysis with anti-LAT antibody. This result
strongly suggests that CD2- and CD3-lipid raft associations are
independent of one another. Additional evidence supporting this view
comes from the observation shown in Fig. 1 (lane 6) that
anti-CD3
mAb did not induce association of CD2 with the
detergent-insoluble fraction.
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Fig. 5.
CD2-lipid raft association is independent of
CD3-raft recruitment and TCR signaling. A, 1 × 108 activated human T cells were stimulated with either a
combination of anti-T112 + anti-T113 mAbs
(CD2XL) or anti-CD3 mAb 2Ad2 (CD3XL)
at 37 °C for 10 min. Total cell lysates were prepared using 0.2%
Triton X-100 (TX-100) and subjected to sucrose gradient
separation of lipid rafts. Only raft fractions were run on
SDS-polyacrylamide gel and Western-blotted (WB) for CD3
.
Subsequently, the membranes were stripped and reblotted (RB)
with anti-CD2 or anti-LAT polyclonal antibodies. B, 8 × 107 J77 cells were pretreated with PP2 at 37 °C for
10 min or left untreated (
). Anti-T112 + anti-T113 antibodies were used to stimulate both cells at
37 °C for 15 min. Total cell lysates were then fractionated on
sucrose gradients, and raft fractions were subjected to Western
blotting with anti-CD2 mAbs and reblotting with anti-LAT
antibodies.
25 and CD2
7 (41), which retain only 25 and 7 amino acids, respectively, of the 117 CD2 tail residues (Fig. 6A). Surprisingly, CD2
25
and CD2
7 translocated to the DIG fraction after anti-CD2 mAb
cross-linking (Fig. 6B), even though these truncations
eliminate the components of the CD2 tail that interact with known
signaling molecules (43, 44). Thus, the CD2 tail is not responsible for
the inducible DIG association, raising the possibility that either the
CD2 extracellular domain or transmembrane region might be necessary. We
therefore created chimeric molecules consisting of the CD2
extracellular segment fused with the transmembrane region of a non-raft
resident molecule, CD71 (TfR). Prior studies showed that CD71 was
excluded from lipid rafts using biochemical methods (17, 22) as well as
microscopic observations (23). To this end, we cloned the TfR
transmembrane domain by reverse transcription-polymerase chain reaction
and fused it to a human CD2 extracellular domain, with
(TfR25) or without (TfRTM) appending the
membrane-proximal 25 amino acid residues of the CD2 cytoplasmic tail.
Subsequently, stable transfectant cell lines were established using the
155.16 mouse T cell hybridoma cells as a recipient. As shown in Fig.
6C, the CD2 transmembrane region was not responsible for
CD2-DIG translocation: TfR25 and wild-type CD2
(W33) molecules were equivalently translocated to DIG upon
anti-CD2 mAb cross-linking. In the case of the TfRTM
variant, translocation was also observed, although the reduced surface
CD2 expression of the TfRTM variant yielded only a 3.0-fold
increase compared with TfR25 (9.2-fold increase). As
an independent means of verifying these results, we made chimeric
molecules using a mutant LAT transmembrane domain in lieu of that of
CD71. In this mutant LAT segment, two cysteines were mutated to serine
to abolish the palmitoylation of the LAT transmembrane domain,
responsible for its localization in lipid rafts (53). These results
also showed that the lipid raft association of the CD2 chimeric
molecule upon CD2 cross-linking was not impaired (data not shown).
Therefore, we conclude that the CD2 transmembrane domain is not
required for its relocation to lipid rafts.
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Fig. 6.
Domains of CD2 responsible for lipid raft
recruitment. 155.16 T cells transfected with wild-type
(W33) (41) or mutant CD2 molecules were either cross-linked
by a combination of anti-T112 + anti-T113
ascites (+) or left untreated ( ). Lipid raft fractions were separated
on sucrose gradients, and equal amounts were loaded on
SDS-polyacrylamide gel and then subjected to Western blot
(WB) analysis with anti-CD2 mAbs. The same membrane was
stripped and reblotted (RB) with anti-LAT antibodies to
ensure equivalent loading of each raft fraction. A, sequence
alignment of the CD2 transmembrane and cytoplasmic membrane-proximal
regions. The thick line represents the transmembrane domain
(TM). The two arrows indicate the
unique membrane-proximal cysteine residues in rodent CD2. The
broken lines represent the constructions used in
B. Residues that are identical in at least three of the
homologs are boxed. B, analysis of wild-type CD2
(W33), CD2 truncated after residue 25 in its tail
(
25), and CD2 truncated after residue 7 in its
tail (
7). The results shown are representative
of four experiments. C, analysis of CD2 mutants in which the
transmembrane segment was swapped with the transferrin receptor
transmembrane region with (TfR25) or without
(TfRTM) 25 amino acids of the CD2 tail appended. For
quantitation purposes, the relative intensity of unstimulated cells is
set at 1.0. D, analysis of glycan-deficient CD2 mutants. CD2
molecules with a single N117Q or N126Q mutation or a double N117Q/N126Q
glycan mutation are shown. The experiment was repeated three times with
similar results.
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Fig. 7.
CD2 interaction with CD58 during cell-cell
conjugation is sufficient to recruit CD2 to lipid rafts.
A, 1 × 108 J77 cells were mixed with CHOP
or CHO58 cells (5:1) and incubated at 37 °C for 5 min. The cell
lysates were made and subjected to sucrose gradient separation. Lipid
raft fractions (fraction 3 (Frac3)) and soluble fractions
(fraction 10 (Frac10)); one-fifth the amount of fraction 3)
were resolved by SDS-PAGE and Western-blotted (WB) with
anti-CD2 M32B polyclonal antisera. The membrane was stripped and
reblotted (RB) with anti-LAT antibodies to ensure the equal
loading of raft fractions. Numbers under the upper
panel represent the relative intensity of each band. Shown is a
blot representative of four individual experiments. B,
1 × 108 activated human T cells were treated
identically as described for A, except that the CHO
transfectant bearing the mutant CD58 molecule K87A known to disrupt CD2
binding (CHO58K87A) (68) was used as a negative control. This
experiment was repeated three times.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor I
(26, 27), a heterotetrameric complex expressed on basophils and mast cells. Ligand-engaged IgE receptors, but not unaggregated receptors, partition into lipid rafts (26). This interaction was found to be very
sensitive to detergent extraction, so only in 0.025% Triton X-100
could this association be observed (26). A similar behavior was
identified for the TCR in T lymphocytes (17, 22). Upon TCR ligation or
antibody-mediated cross-linking, an increased amount of TCR was found
in lipid raft fractions (22). Moreover, phosphorylated
-chain as
well as phosphorylated ZAP70 and phospholipase C
1 were also observed
in rafts (17), showing that the inducible TCR-raft translocation
correlates with co-association of known signaling molecules. As with
Fc
receptor I, the association was sensitive to the specific
detergent and its concentration (8, 22). Thus, weaker detergents such
as Brij 58 preserved TCR-raft interaction, whereas 1% Triton X-100 or
Nonidet P-40 did not (22). Similar to the TCR, the B cell receptor was
also shown to translocate into lipid rafts upon antibody cross-linking
(24, 25).
receptor I, being stable
even using 1% Triton X-100 extraction conditions. This distinction in
raft association strength implies that the mechanisms involved in their
respective association could be different. Interestingly, another
monomeric lymphocyte surface protein, CD20, which is expressed on B
cells, shows very similar properties to CD2 in terms of inducible
association following antibody cross-linking and detergent stability
(71). Notwithstanding, CD20 and CD2 are structurally unrelated.
mAb cross-linking activates multiple proximal TCR
signaling molecules such as ZAP70 and p56lck as evident by
their phosphorylation. Activated p56lck and LAT, a substrate of
ZAP70, are both resident lipid raft proteins. p56lck and ZAP70
bind to immune receptor tyrosine-based activation motifs in the
cytoplasmic tail of the CD3
, CD3
, CD3
, and CD3
subunits of
the TCR. These interactions can recruit the TCR to rafts. In support of
the notion that signal transduction is responsible for TCR relocation
to rafts, it was reported that the phospho-TCR
-chain can interact
with the raft resident p59fyn SH2 domain, suggesting that
localization of Src family kinases is also important (15).
7 truncation, and yet this molecule redistributes to rafts like wild-type CD2. Stimulating cells with anti-CD3
mAb cross-linking fails to recruit CD2 to rafts (Fig. 5A), further indicating that the mechanisms behind TCR- and
CD2-raft recruitment are different.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Harald von Boehmer, Linda Clayton, and Elena Tibaldi for critical comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI21226.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Lab. of Immunobiology,
Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.:
617-632-3412; Fax: 617-632-3351; E-mail: ellis_reinherz@ dfci.harvard.edu.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M009852200
2 Polymerase chain reaction primer sequences are available upon request.
3 H. Yang and E. L. Reinherz, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
TCRs, T cell
receptors;
pMHC, peptide bound to a major histocompatibility complex
molecule;
APC, antigen-presenting cell;
DIG, detergent-insoluble glycolipid-enriched microdomain;
IL, interleukin;
mAb, monoclonal antibody;
-MCD, methyl-
-cyclodextrin;
TBS, Tris-buffered saline;
MES, 4-morpholineethanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis;
TfR, transferrin receptor;
ERK, extracellular signal-regulated kinase;
CHO, Chinese hamster ovary;
EPO, erythropoietin.
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