This information is current as
of June 18, 2017.
Recombinant Anti-CD4 Antibody 13B8.2
Blocks Membrane-Proximal Events by
Excluding the Zap70 Molecule and
Downstream Targets SLP-76, PLC γ1, and
Vav-1 from the CD4-Segregated Brij 98
Detergent-Resistant Raft Domains
Myriam Chentouf, Soufiane Ghannam, Cédric Bès, Samuel
Troadec, Martine Cérutti and Thierry Chardès
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 179:409-420; ;
doi: 10.4049/jimmunol.179.1.409
http://www.jimmunol.org/content/179/1/409
The Journal of Immunology
Recombinant Anti-CD4 Antibody 13B8.2 Blocks
Membrane-Proximal Events by Excluding the Zap70 Molecule
and Downstream Targets SLP-76, PLC␥1, and Vav-1 from the
CD4-Segregated Brij 98 Detergent-Resistant Raft Domains1
Myriam Chentouf,* Soufiane Ghannam,* Cédric Bès,2* Samuel Troadec,* Martine Cérutti,†
and Thierry Chardès3*
I
n 1972, Singer and Nicolson proposed a fluid mosaic model
to describe cell membrane organization where supramolecular platforms of proteins and lipids float within the plasma
membrane lipid bilayer. Distinguished from the rest of the membrane by their protein and lipid composition, small-sized (10 –200
nm mean diameter) domains (1), termed membrane rafts, are enriched in lipids containing long-chain saturated fatty acid residues
(mainly sphingomyelin and ceramides) together with cholesterol;
GPI-anchored, palmitoylated, and myristoylated proteins; and several cytoplasmic proteins associated with the inner leaflet of the
*Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche
5236, Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé, Faculté
de Pharmacie, Montpellier, France; and †CNRS Unité Propre de Service 3040, Saint
Christol-Les-Alès, France
Received for publication May 16, 2006. Accepted for publication April 14, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
T.C. was supported by a grant from the Ligue Nationale Contre le Cancer, Comité de
l’Hérault. S.T. held, successively, doctoral fellowships from the Ligue Nationale contre le
Cancer, Comité de l’Hérault, and the Association de Recherche contre le Cancer. M.Ch.
was a recipient of a doctoral fellowship from the Ligue Nationale contre le Cancer,
Comité de l’Hérault. C.B. was a recipient of a postdoctoral fellowship from the Fondation
de France.
2
Current address: Institut de Recherche Pierre Fabre, 5 Avenue Napoléon III, BP
60497, 74164 Saint-Julien en Genevois, France.
3
Address correspondence and reprint requests to Dr. Thierry Chardès, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5236, Centre d’études
d’agents Pathogènes et Biotechnologies pour la Santé, Faculté de Pharmacie, 15 Avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France. E-mail address: [email protected]
www.jimmunol.org
membrane via covalently attached fatty acid residues. These domains correspond to a particular phase of the lipid bilayer, the
liquid-ordered (lo)4 phase (2), floating around in a liquid-disordered phase of glycosphingolipids in the exoplasmic leaflet. The lo
and liquid-disordered phases show distinct behavior when treated
with mild nonionic detergents; such domains from the lo phase are
resistant to Triton X-100, CHAPS, or Nonidet P-40 mainly when
used at low temperature, leading to detergent-resistant membranes
(DRM). The role of DRM in the plasma membrane is still, however, a subject of controversy (3), essentially because DRM obtained by low temperature extraction does not exactly reflect the
raft domains in cell membranes under physiological conditions.
Because of the presence of several receptors or signal-transducing kinases, but also lipids such as ceramides which could act as
second messengers, the DRM platforms have been shown to play
a crucial role in the cell-signaling network which fine tunes various
biological effects. The ability of raft DRM to segregate receptors
provides a mechanism for compartmentalization of signaling components in the plasma membrane, concentrating certain components in membrane rafts and excluding others. On the basis of
these observations, one emerging hypothesis is that the biological
4
Abbreviations used in this paper: lo, liquid ordered; DRM, detergent-resistant membrane; PKC␪, protein kinase C␪; SLP-76, Src homology 2-domain-containing leukocyte protein of 76 kDa; PLC␥1, phospholipase C␥1; Vav-1, p95vav; LAT, linker for
activation of T cells; GM1, ganglioside M1; Cbp/PAG, Csk-binding protein/
phosphoprotein associated with glycosphingolipid; M␤CD, methyl-␤-cyclodextrin;
PBS-T, PBS containing 0.1% Tween 20.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
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The biological effects of rIgG1 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that
prevent NF-␬B nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal
signaling, remain obscure. We support the hypothesis that rIgG1 13B8.2 acts by interfering with the spatiotemporal distribution
of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG1 13B8.2 was found
to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes
at 37°C, together with recruitment of TCR, CD3␨, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and
Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase C␪, but excluded
Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase C␥1, and p95vav.
Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr292 and Tyr319 phosphorylation
occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging
confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred
within 10 –30 s following rIgG1 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The
differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-␬B nuclear translocation and explains how baculovirus-expressed
CD4-CDR3-like-specific rIgG1 13B8.2 acts to induce its biological effects. The Journal of Immunology, 2007, 179: 409 – 420.
410
FIGURE 1. Dot-blot characterization of Brij 98 DRM fractions from
lymphocytes untreated or previously cross-linked by the baculovirus-expressed rIgG1 anti-CD4 Ab 13B8.2. A2.01 CD4⫺ cells and Jurkat and
A2.01 CD4⫹ cells were lysed with Brij 98 detergent at 37°C and segregated into fractions by sucrose density gradient. Fraction 12 represents the
bottom liquid fraction of the tube. Membrane rafts are in fractions 4 – 6 as
they contain ganglioside GM1. Nonraft fractions 11 and 12 were identified
by binding of the transferrin receptor CD71. The CD4 receptor was detected by a goat polyclonal anti-human CD4 Ab. Results are representative
of at least three independent experiments.
Materials and Methods
Cells
Jurkat, Sup-T1, and A2.01-CD4⫹ and -CD4⫺ T cells were grown in RPMI
1640 (Cambrex) supplemented with 10% heat-inactivated FCS (PAA Laboratories), antibiotics (100 U/ml penicillin and 100 ␮g/ml streptomycin;
Sigma-Aldrich), and 2 mM glutamine. Cells were provided by L. Briant
(Centre National de la Recherche Scientifique (CNRS) Unité Mixte de
Recherche (UMR) 5236, Montpellier, France). Primary T lymphocytes
were purified by Histopaque (Sigma-Aldrich) density centrifugation of
blood samples from healthy donors obtained at the Etablissement Français
du Sang (Montpellier, France).
Abs and reagents
Anti-CDR3-like rIgG1 and Fab (rFab) 13B8.2 Abs were expressed in the
baculovirus/insect cell system and purified from culture supernatant by
protein A and G immunoaffinity chromatography, respectively, as previously described (17, 18, 26). Other anti-CD4 mAbs used were directed
against the CDR2-like loop (ST4; Sanofi-Aventis), the CDR3-like loop
(ST40; Sanofi-Aventis), the D2 domain (BF5; Diaclone), and the D4 domain (OKT4; Ortho Biotech). Chinese hamster ovary-expressed anticarcinoembryonic Ag recombinant control IgG1 was a gift from C. Germain
(Institut National de la Santé et de la Recherche Médicale Unité 860, Montpellier, France). Goat polyclonal anti-CD4 Ab was obtained from R&D Systems. Anti-human TCR mAb (IP26) was purchased from BD Biosciences. The
anti-CD3 (UCHT1) and anti-CD28 (CD28.2) mAbs were obtained from Beckman Coulter. The Alexa Fluor 488- and the peroxidase-conjugated cholera
toxin B subunits were purchased from Molecular Probes; Cy3- and Cy5-conjugated Abs came from Jackson ImmunoResearch Laboratories. Rabbit polyclonal Abs to CD71, Syk p70, p56 Lck, Lyn, Zap70, and mAbs directed
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effects of new biotechnological drugs such as therapeutic Abs are
linked to their ability to quantitatively and qualitatively modulate
the clustering of target membrane receptors, signaling kinases, apoptosis-, proliferation-, and cytotoxicity-involved molecules, and
lipids into these raft domains. Concerning the therapeutic Ab antiCD20 rituximab, but also other anti-CD20 Abs, there is a correlation between the ability of these Abs to elicit CD20 translocation
into Triton DRM (4) and their capacity to initiate kinase-dependent
calcium mobilization, apoptosis induction (5), and complementdependent cytolysis (6) of B lymphoma cells. Rituximab also induces ceramide accumulation together with CD20 translocation
into Triton DRM, leading to activation of the ceramide-triggered
signaling pathway, which mediates proliferation inhibition of B
lymphoma cells (7). Lipids from DRM such as ceramide or
cholesterol play a major role in the modulation of apoptosis (8,
9) and cell growth (7) but also influence the outcome of HIV
infection (10). In this case, the virus highjacks raft DRM for
cell entry and further budding, and the CD4 molecule, which is
the primary receptor for virus anchoring, is predominantly localized in the DRM.
CD4 is a 55-kDa glycoprotein expressed on ⬃60% of peripheral
blood T lymphocytes; it activates T lymphocytes by binding to the
nonpolymorphic region of the MHC class II Ags expressed on the
surface of APCs. Engagement of CD4 by therapeutic mAbs triggers various effects actually exploited in cancerology (11), HIV
infection (12), and tolerance induction (13). CD4 epitope recognition by Abs has not been taken into consideration in clinical
studies, except in the case of HIV infection. Nevertheless, geneactivating potential such as NF-AT activation, Ras/protein kinase
C (PKC) pathway inhibition, and proapoptotic potential of antiCD4 mAbs has been associated with recognition of different CD4
epitopes (14, 15). Biological effects (6) and, above all, different
abilities to relocalize CD20 in DRM (16) are also shown to be
related to epitope recognition of anti-CD20 Abs. We evaluated a
recombinant Ab (17, 18) derived from the 13B8.2 murine antiCD4 mAb, directed against the CDR3-like loop of CD4; this loop
has not been fully exploited as a target by clinical Abs, mainly
directed against other CD4 regions (11–13, 19, 20). This baculovirus-expressed recombinant Ab (rIgG1) inhibits HIV replication at a post-gp120-binding step (17, 18, 21) and induces
complement-mediated lysis, Ab-dependent cell cytotoxicity,
and growth arrest of T lymphoma cells (22). The biological
effects of rIgG1 13B8.2 are partly due to signals that prevent
NF-␬B nuclear translocation (23); ERK activation (24); and
NF-AT, NF-␬B, and AP-1 binding to the IL-2 gene promoter
(25). However, precise mechanisms of action at the early level
of membrane proximal signaling to explain how baculovirusexpressed CD4-CDR3-like-specific rIgG1 13B8.2 acts to induce
its biological effects remain obscure. We support the hypothesis
that rIgG1 13B8.2 acts by interfering with the distribution of
signaling or receptor molecules inside DRM platforms.
With this perspective in mind, we treated target Jurkat lymphoma cells with rIgG1 13B8.2 and demonstrated for the first time
that rIgG1 13B8.2 acts by inducing an accumulation/retention of
the CD4 molecule inside polyoxyethylene-20 ether Brij 98 DRM
extracted at 37°C together with exclusion of the Zap70 kinase and
its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa (SLP-76), phospholipase C␥1 (PLC␥1), and
p95vav (Vav-1). These results suggest that analysis of the lipidprotein rheostat inside Brij 98 DRM following treatment with biotechnological drugs could explain how these molecules induce
their biological effects.
ANTI-CD4 13B8.2-INDUCED CD4/Zap70 RAFT PARTITIONING
The Journal of Immunology
411
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FIGURE 2. rIgG1 anti-CD4 Ab 13B8.2 cross-linking of Jurkat T cells induced the redistribution of CD4 into the membrane rafts and the exclusion of
the Zap70 and downstream targets SLP-76, Vav-1, and PLC␥1. A, Cells were cross-linked in solution with rIgG1 13B8.2 or with an irrelevant control IgG1
Ab, and sucrose gradient fractions were Western blotted with goat-polyclonal anti-CD4 Ab; anti-TCR mAb, anti-CD3␨, and anti-phosphorylated CD3␨
mAbs; rabbit polyclonal Abs directed against CD71, Zap70, p56 Lck, Lyn, and Syk p70; and anti-LAT, -Cbp/PAG, -LIME, -SLP-76, -Vav-1, -PLC␥1, and
-PKC␪ Abs. Data are representative of three experiments. B, Raft fraction 4, nonraft fraction 12, and insoluble sediments from control IgG1- or 13B8.2treated cells were immunoblotted with Abs directed to CD4, Zap70, SLP-76, Vav-1, and PLC␥1.
against nonphosphorylated p16 (6B10.2) and the phosphorylated p21/p23
CD3␨ chain (CA415.9A) were obtained from Santa Cruz Biotechnology. Rabbit polyclonal Abs directed against Tyr319- and Tyr493-phosphorylated Zap70,
Vav-1, PLC␥1, and PKC␪ were purchased from Cell Signaling; rabbit polyclonal Abs to Tyr292-phosphorylated Zap70 and to SLP-76 were obtained from
BD Biosciences. Rabbit anti-linker for activation of T cells (LAT) and antiLIME Abs were obtained from Upstate and Abcam, respectively. Csk-binding
protein/phosphoprotein associated with glycosphingolipid (Cbp/PAG)-specific
mAb was purchased from Alexis Biochemicals. Anti-phosphotyrosine
mAb 4G10 was a gift from C. Gongora (Centre Régional de Lutte contre le
Cancer Val d’Aurelle, Montpellier, France). Peroxidase-conjugated antirabbit, anti-mouse, and anti-goat IgG (Sigma-Aldrich) were used as secondary
Abs when necessary. Unconjugated polyclonal anti-human IgG Ab, polyoxyethylene-20 ether detergent Brij 98, methyl-␤-cyclodextrin (M␤CD), and cytochalasin D were purchased from Sigma-Aldrich. Soluble PP2 4-amino-5(4chlorophenyl-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine) was from Merck
Biosciences. Baculovirus-expressed soluble recombinant gp120 from HIV
was a gift from F. Veas (Institut de Recherche pour le Développement,
Unité de Recherche 34, Montpellier, France) (27).
Lymphocyte treatment by Abs and Brij 98-DRM isolation
A total of 1 ⫻ 108 T cells was treated for 10 min with 20 ␮g/ml rIgG1
13B8.2 or rFab 13B8.2 or other anti-CD4 Abs diluted in complete medium.
412
ANTI-CD4 13B8.2-INDUCED CD4/Zap70 RAFT PARTITIONING
In some experiments, rIgG1 13B8.2-pretreated cells were further stimulated with a mixture of anti-CD3 (1 ␮g/ml) and anti-CD28 (5 ␮g/ml) mAbs
for 30 min. In the gp120 experiment, cells were preincubated for 30 min
with recombinant gp120 (10 ␮g/ml) before anti-CD4 treatment. For the
hyper-cross-linking experiment of rIgG1 13B8.2, the cells were further
incubated for 20 min at room temperature with rabbit anti-human IgG
polyclonal Ab. After washing in 160 mM PBS (pH 7.4), cell lysis was
performed at 37°C for 30 min in 1% Brij 98 detergent diluted in TNE buffer
(25 mM Tris-HCl (pH 7.5); 150 mM NaCl; and 5 mM EDTA) containing
1 mg/ml enzyme inhibitors (complete EDTA-free mixture of antiproteases;
Roche). Cell lysates were mixed with an equal volume of 80% sucrose in
TNE plus inhibitors, overlaid with 6.5 ml of 30% and 3.5 ml of 5% sucrose
in TNE plus inhibitors, and then centrifuged at 200,000 ⫻ g for 20 h. From
the top of the gradients, 12 1-ml fractions were collected on ice and numbered from 1 to 12. The protein in each fraction was quantified by using the
micro Bradford Protein Assay kit (Pierce).
Lymphocyte pretreatment by inhibitors
A total of 1 ⫻ 108 cells was preincubated with 10 mM M␤CD diluted in
PBS for 30 min at 37°C to extract cholesterol, or with 20 ␮M cytochalasin
D to prevent the cytoskeletal actin polymerization, or with 10 ␮M soluble
PP2 to induce dephosphorylation. Cells were washed with PBS to remove
inhibitors and treated with Abs as described above. Absence of inhibitor
toxicity was confirmed by trypan blue exclusion in pretreated and untreated
cells.
Dot-blot analysis of ganglioside M1-enriched Brij 98 DRM
A nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech)
was spotted with 2 ␮g from each gradient fraction. The membrane was
blocked for 1 h at room temperature with 5% semiskimmed milk in PBS
containing 0.1% Tween 20 (PBS-T). Ganglioside M1 (GM1) detection in
Brij 98 DRM was performed by adding a 1/1000 solution of peroxidaseconjugated cholera toxin B subunit and incubating for 1 h at room temperature. Nonraft fractions were detected following a 1-h incubation of a
1/1000 dilution of anti-CD71 polyclonal Ab. CD4 was probed by using a
1/3000 solution of polyclonal goat anti-CD4 Ab. After three washes in
PBS-T, the appropriate peroxidase-conjugated secondary Ab was added
and the membranes were incubated for 1 h at room temperature for detection of CD4 and CD71. After washing in PBS-T, dot blots were finally
developed using the ECL Western Blotting Detection kit (Amersham Pharmacia Biotech).
SDS-PAGE and Western blot
Forty micrograms of protein lysate from each gradient fraction were separated by 12% SDS-PAGE under reducing conditions and electrophoretically transferred to Immobilon P (Bio-Rad). The membranes were blocked
with 5% semiskimmed milk in PBS-T for 1 h at 37°C. After washing in
PBS-T, membranes were incubated with an appropriate dilution of Abs
directed against CD4, CD71, TCR, p16 CD3␨ chain and its phosphorylated
forms; Syk p70, p56 Lck, and Lyn kinases; Zap70 and its phosphorylated
forms; LAT, SLP-76, LIME, and Cbp/PAG adaptors; and Vav-1, PLC␥1,
and PKC␪ for 2 h at room temperature. The membranes were then washed
three times with PBS-T and revelation was performed for 1 h at room
temperature with secondary peroxidase-conjugated anti-rabbit Ab (1/3000
dilution), anti-mouse Ab (1/1000), or anti-goat Ab (1/1000), as appropriate.
After three washes, all Western blots were developed using the ECL Western Blotting Detection kit (Amersham Pharmacia Biotech).
Confocal microscopy
A total of 1 to 5 ⫻ 105 cells, treated with 13B8.2 Ab for 10 min, was
washed and incubated in PBS for various times before fixing in 3% paraformaldehyde in PBS for 15 min at room temperature and then permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After washing
in PBS containing 2% BSA (PBS-BSA), the cells were incubated for 1 h
at room temperature with the Alexa Fluor 488-conjugated cholera toxin B
subunit, anti-CD4 rIgG1 13B8.2, or polyclonal anti-Zap70 Ab diluted in
PBS-BSA and then washed. For CD4 and Zap70 detection, the cells were
incubated for 45 min at room temperature in the dark with secondary Cy5and Cy3-conjugated Abs, respectively. After washing, bound cells were
settled onto polylysine-coated slides and analyzed with a Zeiss LSM 510
laser scan confocal microscope (P. Travo, Montpellier RIO Imaging,
CNRS UMR 5237, Montpelier, France) for visualization. Colocalization
assays were analyzed by excitation of the corresponding fluorochromes on
the same section of the permeabilized or nonpermeabilized samples. Negative controls lacking the primary Ab showed no staining. Percentage of
colocalization was obtained by using Imaris software (Bitplane).
Results
Recombinant anti-CD4 Ab 13B8.2 cross-linking of Jurkat T cells
redistributes CD4 into the membrane rafts but excludes the
Zap70 molecule
Dot-blot preliminary experiments were performed on fractions extracted at 37°C with the polyoxyethylene mild detergent Brij 98
from T cell lines A2.01 CD4⫺, Jurkat CD4⫹, and A2.01 CD4⫹. As
shown in Fig. 1, GM1 detection, which identifies DRM raft fractions, was mainly observed in fractions 4 – 6, whereas CD71 binding was evidenced in fractions 11 and 12, identifying nonraft fractions. This pattern was obtained with the three cell lines tested, but
with slightly different intensities. Without 13B8.2 Ab cross-linking, the CD4 molecule was demonstrated either inside the Brij 98
DRM or in the nonraft fraction from Jurkat CD4⫹ and A2.01
CD4⫹ cells but was not detected in fractions from A2.01 CD4⫺
cells, thus demonstrating the binding specificity (Fig. 1). Baculovirus-expressed CD4-CDR3-like specific rIgG1 13B8.2 treatment
completely redistributed the CD4 molecule inside the Brij 98
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FIGURE 3. rIgG1 anti-CD4 Ab 13B8.2 cross-linking of either A2.01 CD4⫹ and Sup-T1 cell lines or primary T cells similarly induced the translocation of CD4
into the membrane rafts and the exclusion of the Zap70 molecule. Cells were cross-linked in solution with rIgG1 13B8.2 or with an irrelevant control IgG1 Ab,
and sucrose gradient fractions were immunoblotted (A) with a goat polyclonal anti-CD4 Ab or (B) with a rabbit polyclonal Ab directed against Zap70.
The Journal of Immunology
413
DRM, whereas no CD4 binding was observed in fractions extracted from A2.01 CD4⫺ cells.
In an attempt to link 13B8.2-dependent CD4 translocation inside
Brij 98 DRM with the downstream signaling pathways, we examined the distribution of CD4, TCR, CD3␨ chain, various kinases,
and adaptor proteins among raft/nonraft fractions following Ab
treatment (Fig. 2A). First, raft and nonraft fractions were similarly
identified as shown in Fig. 1 by GM1 and CD71 detection, and raft
compartmentalization of CD4 upon rIgG1 13B8.2 treatment was
confirmed (Fig. 2A). Western blot analysis revealed that rIgG1
13B8.2 cross-linking of Jurkat T cells completely redistributed
the Zap70 kinase and downstream targets SLP-76, PLC␥1, and
Vav-1 in the nonraft fraction. These molecules were not detected in the insoluble sediments (Fig. 2B), neither following
control IgG1 nor following anti-CD4 rIgG1 13B8.2 treatment.
In addition, CD4/Zap70 redistribution was also observed when
A2.01 CD4⫹ and Sup-T1 cell lines and when primary periph-
eral T lymphocytes from a healthy donor were treated with
rIgG1 13B8.2 (Fig. 3).
In addition, TCR, CD3␨ chain p16, other kinases such as p56
Lck, Lyn, and Syk p70, and also adaptor proteins such as LAT,
Cbp/PAG, and PKC␪ were located inside Brij 98 DRM upon
rIgG1 13B8.2 treatment (Fig. 2A) as did CD4. In contrast, equal
partitioning between raft/nonraft fractions was observed for
these molecules upon treatment with a control IgG1 Ab, except
for LIME, which remained in the raft fraction of cells either
treated with the control Ab or with Ab 13B8.2. Phospho-CD3␨
chain, only detected in the nonraft fractions upon Jurkat treatment with control IgG1 Ab, was not evidenced following cell
cross-linking by anti-CD4 rIgG1 13B8.2. Taken together, these
results indicate that the baculovirus-expressed CDR3-like specific rIgG1 13B8.2 acts by excluding the Zap70 and downstream
molecules SLP-76, PLC␥1, and Vav-1 from the CD4-segregated raft machinery.
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FIGURE 4. Displacement of Zap70 from the 13B8.2-induced CD4-segregated raft signaling platform from native Jurkat cells occurred concomitantly
with its modulation of phosphorylation. A, Cells were treated with control IgG1 Ab or rIgG1 anti-CD4 Ab 13B8.2 alone. Sucrose gradient fractions were
immunoblotted with rabbit polyclonal Abs directed against Zap70 and phosphorylated-Tyr292, -Tyr319, or -Tyr493 Zap70. Data are representative of three
experiments. B, Cells were pretreated with control IgG1 Ab or rIgG1 anti-CD4 Ab 13B8.2 and stimulated with CD3/CD28 mAbs. C, Whole cell lysates
of untreated or Ab-treated cells were immunoblotted with anti-phosphotyrosine mAb 4G10.
414
ANTI-CD4 13B8.2-INDUCED CD4/Zap70 RAFT PARTITIONING
Displacement of Zap70 from the 13B8.2-induced CD4segregated raft signaling platform occurred concomitantly with
its modulation of phosphorylation
FIGURE 6. rIgG1 13B8.2-induced
CD4/Zap70 partitioning is epitope
dependent but does not need Ab hyper-cross-linking. Before Brij 98
DRM preparation, Jurkat T cells were
treated (A) with control IgG1 Ab or
anti-CD4 Abs rIgG1 13B8.2, ST40,
ST4, BF5, and OKT4, which are directed against various epitopes on the
CD4 molecule; or (B) with rIgG1
13B8.2 followed by a rabbit polyclonal anti-human IgG Ab for Ab hyper-cross-linking or rFab 13B8.2
alone. CD4 detection by Western blot
was performed by using a goat polyclonal anti-CD4 Ab, and Zap70 binding was visualized by a specific rabbit
polyclonal Ab.
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FIGURE 5. rIgG1 13B8.2 treatment of gp120-cross-linked Jurkat T
cells did not altered CD4/Zap70 differential partitioning but differently affects gp120-induced Zap70 phosphorylation. Cells were (A) left untreated
or (B) incubated with gp120 or (C) preincubated with gp120 and treated by
rIgG1 13B8.2. Cells were further lysed with Brij 98 detergent, and the
fractions were separated by sucrose density centrifugation. Following
PAGE-SDS and immunoblotting, CD4 binding was assessed by probing
with a goat polyclonal anti-CD4 Ab, and Zap70 was visualized by a specific rabbit polyclonal Ab. Fractions from treated Jurkat cells were detected
by rabbit polyclonal Abs directed against phosphorylated-Tyr292, -Tyr319,
or -Tyr493 Zap70. Data are representative of three experiments.
Early phosphorylation events on tyrosine residues in the Zap70
kinase are pivotal for its enzymatic activation and subsequent stimulation of downstream signaling pathway. Together with the Abinduced Zap70 exclusion from the CD4-segregated Brij 98 DRM,
we hypothesized that the phosphorylation profile of the kinase was
modulated upon rIgG1 13B8.2 cross-linking. Because numerous
tyrosine phosphorylation sites can be affected on Zap70, we focused on the three most critical residues, two of them phosphoTyr319 and phospho-Tyr493, being known to favor CD3/TCR-mediated immune response, and the third, phospho-Tyr292, known to
inhibit the immune response by targeting Zap70 to Cbl-mediated
ubiquitination/degradation. As demonstrated in Fig. 4A, 13B8.2
cross-linking of Jurkat T cells led to phosphorylation of the Tyr292
residue in the Zap70 kinase redistributed in the nonraft fraction. In
contrast, no Tyr292-specific phosphorylation was evidenced upon
binding of a control IgG1 Ab. Simultaneously, activating phosphorylation of Tyr319 on the Zap70 kinase was inhibited upon
rIgG1 13B8.2 cross-linking. Finally, no phosphorylation of activating Tyr493 occurred either following control IgG1 treatment or
upon anti-CD4 13B8.2 binding (Fig. 4A). These results suggest
that the baculovirus-expressed CDR3-like specific rIgG1 13B8.2
acts by favoring inhibiting phosphorylation of Tyr292 and blocking
activating phosphorylation of Tyr319 together while excluding the
Zap70 molecule from the CD4-segregated raft machinery of Jurkat
T cells.
To assess whether Zap70 compartmentalization and its phosphorylation pattern were also affected when both Ag receptor and
CDR3-like loop are engaged, we pretreated Jurkat T cells with
rIgG1 13B8.2 before stimulation with the anti-CD3 and anti-CD28
mAbs. As shown in Fig. 4B, Zap70 was evidenced in the nonraft
fraction upon CD3/CD28 stimulation alone together with the induction of activating phosphorylation of Tyr319 and Tyr493
whereas inhibiting phosphorylation of Tyr292 was not observed.
Activating phosphorylation of Tyr319, observed upon CD3/CD28
stimulation, was affected by a pretreatment with 13B8.2 Ab (Fig.
4B). By separating whole lysates from CD3/CD28-stimulated cells
pretreated or not with rIgG1 13B8.2 on 8% PAGE-SDS, we further
assessed tyrosine phosphorylation of multiple proteins, essentially
located in a molecular mass range for Zap70, SLP-76, and Vav-1.
The Journal of Immunology
415
rIgG1 13B8.2 treatment globally increased general tyrosine phosphorylation of proteins in the 64 –98 kDa range (Fig. 4C).
rIgG1 13B8.2 treatment of gp120-cross-linked Jurkat T cells did
not alter CD4/Zap70 differential partitioning but differentially
affects gp120-induced Zap70 phosphorylation
13B8.2-induced CD4/Zap70 differential partitioning is epitope
dependent but does not require Ab hyper-cross-linking
Therapeutic anti-CD20 Abs showed a marked difference in their
ability to translocate CD20 inside Triton DRM, depending on
epitope recognition (6, 16), such an observation being correlated
with their capacity to induce biological effects (6, 16). We used
five anti-CD4 Abs, directed against various epitopes on the CD4
molecule, to assess whether Ab-induced CD4/Zap70 segregation
inside/outside Brij 98 DRM is epitope dependent (Fig. 6A). As
demonstrated, cross-linking of Jurkat T cells by Abs 13B8.2 and
ST40, directed against the CDR3-like loop on the D1 domain of
CD4, induced CD4 partitioning inside raft fractions, whereas control IgG1 did not. In contrast, other anti-CD4 Abs tested, either
directed against the CDR2-like loop on the D1 domain or which
bound to the D2 or D4 domain of CD4, did not modify CD4
raft/nonraft distribution, similarly to the control Ab (Fig. 6A). Anti-CDR3-like Ab 13B8.2 translocated Zap70 inside the nonraft
fraction, whereas neither of the anti-CD4 Abs, directed against
other epitopes on the CD4 molecule, nor a control Ab affected
Zap70 raft/nonraft distribution (Fig. 6A).
In addition, the need for Ab hyper-cross-linking for the efficiency of therapeutic Abs is a subject of debate (6, 16). rIgG1
13B8.2 hyper-cross-linking by secondary anti-human IgG did not
enhance CD4/Zap70 raft segregation from T cells, as confirmed by
the fact that rFab 13B8.2 similarly induced complete CD4 translocation inside Brij 98 DRM and subsequent Zap70 partitioning
inside the nonraft fraction (Fig. 6B). These results indicate that the
effect of the baculovirus-expressed CDR3-like specific rIgG1
13B8.2 on CD4 segregation into rafts is dependent on the CDR3like epitope on the D1 domain of CD4 but does not require hypercross-linking, with formation of an Ab network.
Cholesterol integrity and actin polymerization are critical for
13B8.2-induced CD4 segregation inside Brij 98 DRM
Lipids from DRM such as cholesterol are critical for raft integrity
and can mediate Ab-induced receptor translocation into rafts, lead-
FIGURE 7. Cholesterol integrity and actin polymerization are critical
for rIgG1 13B8.2-induced partitioning. A, Cells were preincubated with the
cholesterol inhibitor M␤CD or with cytochalasin D, which blocks actin
polymerization, before adding rIgG1 13B8.2. Following PAGE-SDS and
immunoblotting, CD4 binding was assessed by probing with a goat polyclonal anti-CD4 Ab. Cells were preincubated (B) with the src inhibitor PP2
alone, which blocks protein phosphorylation, or (C) with PP2 and further
treated with rIgG113B8.2. Following PAGE-SDS of 40 ␮g of protein lysate from each gradient fraction and immunoblotting, CD4 binding was
assessed by probing with a goat polyclonal anti-CD4 Ab and Zap70 binding was visualized by a specific rabbit polyclonal Ab. As controls, quantified sucrose-separated fractions were also characterized with GM1 to
identify the raft fractions and with anti-CD71 Ab to identify the nonraft
fractions.
ing to the modulation of various biological effects (7–10). We
examined whether 13B8.2-induced CD4 raft partitioning is dependent on raft integrity by depleting cholesterol with M␤CD. As
evidenced in Fig. 7A, pretreatment of Jurkat T cells with M␤CD
before rIgG1 13B8.2 treatment, with presumed disruption of Brij
98 DRM, completely redistributed CD4 inside the nonraft fraction, whereas Ab cross-linking, without preincubation with
M␤CD, confirmed CD4 raft compartmentalization (Fig. 7A).
The formation of an immunological synapse requires actin-dependent cytoskeleton restructuring, which can be stopped by an
inhibitor of F-actin polymerization, cytochalasin D. As shown
in Fig. 7A, cytochalasin D pretreatment affected 13B8.2-induced CD4 raft partitioning because CD4 was located in both
the raft and the nonraft fractions, as observed in the absence of
treatment (Fig. 5A) or upon treatment with an irrelevant control
Ab (Figs. 2A and 3A)). In addition, an altered phosphorylation
pattern induced by PP2 preincubation of Jurkat T cells did impact neither on the CD4/Zap70 distribution in raft vs nonraft
fraction of untreated cells (Fig. 7B), nor on the CD4/Zap70
differential partitioning induced by 13B8.2 treatment (Fig. 7C),
suggesting that phosphorylation events do not promote CD4/
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Because Zap70 kinase is involved in the CD3/TCR-mediated signaling pathway, but it is also involved in the gp120-induced signaling pathway following CD4 binding, we examined raft partitioning and the subsequent Zap70 phosphorylation events upon
gp120 cross-linking of Jurkat T cells. As shown in Fig. 5A, equal
CD4 partitioning located between raft/nonraft fractions was observed in untreated Jurkat T cells. In contrast, gp120 binding to
Jurkat T cells redistributed the CD4 molecule inside Brij 98 DRM
and excluded Zap70 from the rafts (Fig. 5B) as did anti-CD4 rIgG1
13B8.2 treatment (Fig. 2A and Fig. 3A). In contrast, the phosphorylation profile was differently affected with regard to that obtained
with 13B8.2-treated cells (Fig. 4A) because the Zap70 Tyr292
residue from the gp120-cross-linked Jurkat T cells was not
phosphorylated, whereas the gp120-cross-linked Jurkat T cells
showed activating phosphorylation of Tyr319 and Tyr493 of the
nonraft-located Zap70 molecule (Fig. 5B). Of interest is that
rIgG1 13B8.2 treatment did not alter the Zap70 nonraft segregation induced upon cross-linking of Jurkat T cells by gp120
(Fig. 5C), but it inhibited the phosphorylation of Zap70 Tyr319
and Tyr493 residues and favored negative regulation of the kinase via phosphorylation of Tyr292 residue.
416
ANTI-CD4 13B8.2-INDUCED CD4/Zap70 RAFT PARTITIONING
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FIGURE 8. Confocal microscopy confirmed the CD4/Zap70 differential partitioning into raft vs nonraft fractions upon cross-linking with rIgG1 13B8.2.
Jurkat cells were treated with irrelevant IgG1 Ab or baculovirus-expressed rIgG1 13B8.2, fixed with paraformaldehyde, permeabilized, and then stained with
Alexa Fluor 488-conjugated cholera toxin B subunit, which binds to GM1-enriched DRM (A and B), or with anti-CD4 (A), or anti-Zap70 polyclonal Abs
(B), followed by appropriate Cy3- and Cy5-conjugates. C, The kinetics of CD4/Zap70 partitioning were observed by confocal microscopy using an
experimental procedure similar to that described above and the GM1/CD4 and GM1/Zap70 clustering was quantified by using Imaris software.
Zap70 compartmentalization. In these experiments, GM1 and
CD71 detection showed a similar binding pattern, in terms of
intensity. Thus, we suggest that the CD4/Zap70 raft partitioning
induced by the baculovirus-expressed CDR3-like specific rIgG1
13B8.2 requires raft integrity, is dependent on actin polymerization, but occurs independently of kinase activity.
The Journal of Immunology
Confocal microscopy visualization confirms CD4/Zap70
differential partitioning which occurs 10 –30 s after rIgG1
13B8.2 cross-linking, reaches a plateau at 1 min, and persists
up to the end of the 1-h experiment
Discussion
Recombinant IgG1 Ab 13B8.2 (rIgG1 13B8.2) has been proposed
as a potential therapeutic agent for the treatment of CD4⫹ malignant diseases because it induces complement-mediated lysis, Abdependent cell cytotoxicity, and growth arrest of T lymphoma cells
(22). We demonstrated in this study that the baculovirus-expressed
rIgG1 13B8.2 Ab acts by causing the accumulation of the CD4
molecule inside the Brij 98-extracted rafts and by excluding the
Zap70 kinase and downstream targets SLP-76, PLC␥1, and Vav-1
from the raft machinery, together with affecting Zap70 phosphorylation, which impairs further downstream activation events.
We favored raft extraction at 37°C with the polyoxyethylene
detergent Brij 98, which led to the purification of a “physiological”
pool of microdomains, as previously demonstrated (1) and recently
confirmed (28). Accordingly, we were able to identify TCRs inside
the raft domains by using this extraction protocol, which is not
possible when detergent with stronger solubilization strength such
as Triton X-100 is used (1). Extraction at 37°C avoided the chilling
step at 4°C used to obtain Triton X-100 DRM, which modifies the
phase behavior of membranes (2). In addition, Brij 98 DRM
showed less aggregation following detergent extraction (1), thus
allowing easier raft characterization. We finally hypothesized that
Brij 98 DRM extraction at 37°C is probably more effective to
detect weak affinity protein associations in rafts, e.g., for the study
of signaling or adaptor proteins, in contrast to Triton X-100 extraction at 4°C (29).
The different raft compartmentalization of CD4 vs Zap70 following treatment with anti-CD4 rIgG1 13B8.2 is important for
subsequent development and longevity of signaling complexes and
for the activation state of downstream molecules. To our knowledge, little data are available concerning a “physical” disconnec-
tion of the Ab-target CD4 molecule with membrane-proximal
Zap70 kinase through dynamically oriented raft segregation. Other
anti-CD4 Abs (30, 31–33) are known to block Zap70 phosphorylation in vitro and in vivo, but neither CD4/Zap70 differential
patching by confocal microscopy nor CD4/Zap70 differential raft
partitioning has been observed. Although no link between murine
parental mAb 13B8.2 and Zap70 kinase has ever been established,
down-regulation of LFA-1-dependent adhesion of Jurkat T cells to
B cells, induced by murine parental mAb 13B8.2 cross-linking,
requires colocalization of p56 Lck, LFA-1, and PI3K, but not Src
homology 2 domain-containing phosphatase SHP-2, in Brij 58
GM3⫹-membrane rafts together with CD4 (34, 35).
Most of the effects induced by 13B8.2 murine mAb, among
which is inhibition of NF-␬B and ERK activation, counteract the
signaling pathways induced by TCR/CD3 engagement (23–25,
36 – 40). This engagement initiates the recruitment and activation
of CD4-noncovalently attached p56 Lck kinase, which phosphorylates the Zap70 kinase (41). This enzymatically activated protein
(42) further phosphorylates the adaptor molecules SLP-76, LAT,
Vav-1, and Grb-2 (43), which form signaling complexes to further
recruit and activate effector enzymes PLC␥1 and PKC (42). rIgG1
13B8.2 acts by decoupling Zap70 and also some of these downstream targets from the CD4-segregated raft machinery of native
Jurkat cells and by modulating the Zap70 phosphorylation profile.
However, the relationship between Ab-induced raft compartmentalization and modulation of phosphorylation remains to be clarified. In our hands, we did not observe Zap70 in raft membranes
from CD3/CD28-stimulated Jurkat T cells, as was previously described following anti-CD3 activation alone (1) and reinforced
when the CD28 costimulation signal was added (44). The low
anti-CD3 concentration we used could in part explain our observation because Ahmed et al. (45), using the same anti-CD3 dosage,
did not demonstrate Zap70 capping. In addition, the immune status
of the cells, i.e., Th1 vs Th2 (46) or naive vs memory cells (47),
also influences membrane reorganization of signaling complexes.
The anti-CDR3-like murine mAbs, including 13B8.2, inhibit
HIV gene expression (48). The 13B8.2-induced effects (23–25, 38,
49, 50) require the cytoplasmic tail of CD4 (51) without the need
of functional p56 Lck or CD45 regulation (49, 52, 53). Mouse
13B8.2 Ab inhibits soluble gp120 binding to soluble and membrane CD4, but fails to block either gp120-bearing virion binding
to CD4 or HIV entry (54), thus suggesting that 13B8.2 and gp120
signaling could be closely related but not equivalent. This argument is correlated with the fact that gp120, like rIgG1 13B8.2,
induces CD4 raft compartmentalization, as demonstrated by us and
others (55), but preferentially triggers phosphorylation of signalactivating Zap70 Tyr319 and Tyr493 residues (41, 42, 56), rather
than phosphorylating the signal-inhibiting Zap70 Tyr292 residue
(57), which we observed upon rIgG1 13B8.2 binding. rIgG1
13B8.2 drives the gp120-induced phosphorylation pattern toward a
Zap70-negative regulation pathway, explaining why anti-CD4
13B8.2 Ab treatment induces inhibition of NF-␬B activation and
transcription inhibition of the viral genome, whereas gp120 binding leads to NF-␬B activation and productive transcription of the
viral genome in the case of HIV infection (23). Of great interest is
that Zap70 is required in infected donor cells for efficient cell-tocell HIV transmission to recipients and for formation of the virological synapse (58).
rIgG1 13B8.2 cross-linking of Jurkat T cells does not induce
Zap70 Tyr493 phosphorylation in the activation loop of Zap70,
which is known to increase TCR/CD3-mediated immune responses (42), probably because DRM-located upstream of p56
Lck, known to specifically phosphorylate Tyr493 residue on the
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Confocal microscopy confirmed the CD4/Zap70 differential partitioning into raft vs nonraft fractions upon cross-linking with rIgG1
13B8.2. Jurkat cells were treated with an irrelevant IgG1 Ab or the
rIgG1 13B8.2, fixed with paraformaldehyde, permeabilized, and
then stained with the Alexa Fluor 488-conjugated cholera toxin B
subunit, which binds to GM1-enriched DRM, or with anti-CD4 or
anti-Zap70 polyclonal Abs followed by appropriate Cy3 and Cy5
conjugates. CD4 (Fig. 8A) and Zap70 (Fig. 8B) were evenly distributed upon treatment of Jurkat cells with irrelevant control IgG1
Ab, colocalized with GM1 but also detected outside GM1⫹-enriched regions. Preferential copatching of GM1 and CD4 was observed when cells were treated with rIgG1 13B8.2 (Fig. 8A); this
was not the case for GM1 and Zap70 in 13B8.2-cross-linked cells
(Fig. 8B). Zap70 stained at sites that are not coincident with GM1enriched areas. We examined up to 1000 cells from several separate areas of 50 –100 cells each, several times, in three independent
experiments and obtained similar results. As shown and quantified
in Fig. 8C, the kinetics of CD4/Zap70 differential partitioning occurred rapidly (10 s) upon Ab treatment. An increase in CD4/GM1
clustering, with ⬃70 – 80% colocalization, was observed 1 min
after cross-linking, whereas a decrease in GM1/Zap70 clustering,
with only 10 –20% residual colocalization, was evidenced at the
same time (Fig. 8C). This inversely different clustering was observed until the end of the experiment (1 h). These kinetics confirm
the differential partitioning of CD4/Zap70 and demonstrate that
these events occur rapidly but are not transient and persist for at
least 1 h.
417
418
D1 domain of CD4 (63). Epitope specificity governing raft localization and further biological effects have mainly been defined for
anti-CD20 reagents (5, 6, 16). It would be interesting to compare
the epitope specificity of anti-CD4 Abs YNB.46 (30), KT6,
YTS177, and YTA3.1 (31, 32), all of which are known to induce
inhibition of Zap70 phosphorylation, with the CDR3-like epitope
specificity (residues Glu87 and Asp88 in the D1 domain of CD4) of
Ab 13B8.2 (64), which reorganizes Zap70 outside the rafts and
modulates its phosphorylation profile. Concerning the anti-CD4
Ab OKT4, directed against the D4 domain, we were not able, using
our experimental procedure, to induce CD4 accumulation/retention
into 37°C-extracted Brij 98 DRM upon OKT4 binding without
anti-mouse hyper-cross-linking, whereas Fragoso et al. (33)
showed an increase in the amount of CD4 together with LAT and
p56 Lck in 4°C-Triton DRM upon OKT4 cross-linking, followed
by anti-mouse hyper-cross-linking. Residual CD4, LAT, and p56
Lck molecules were still observed upon OKT4 binding/hypercross-linking in the nonraft fraction (33), whereas rIgG1 13B8.2
treatment, without hyper-cross-linking, completely localized CD4
into Brij 98 DRM together with LAT and p56 Lck proteins. These
results raise several questions concerning the influence of the detergent (65), the temperature of extraction, and hyper-cross-linking
requirement (5), not yet resolved, but of critical importance to
ascertaining the raft “physiology” (3). In our case, the fact that no
hyper-cross-linking was necessary for rIgG1 13B8.2-induced raft
partitioning correlates with the fact that the monovalent baculovirus-expressed rFab 13B8.2 is sufficient to induce CD4 accumulation/retention in Brij 98 DRM. Although we cannot rule out the
possibility of Ab aggregates, our observation emphasizes the fact
that targeting of two adjacent epitopes by rIgG1 13B8.2 is not
necessary for CD4 clustering inside rafts.
Ceramides and cholesterol are critical for the maintenance and
the dynamics of membrane rafts and CD4 localization in raft domains is regulated by posttranslational lipid modifications (66).
We demonstrated that cholesterol depletion before rIgG1 13B8.2
cross-linking affects subsequent Zap70/CD4 differential partitioning into Brij 98 DRM. The integrity of Brij 58 or Triton DRM,
together with CD20 translocation, Ca2⫹ mobilization, and apoptosis induced by anti-CD20 Abs, is compromised by cholesterol depletion (5). Raft distribution induced by other Abs has been shown
to be impaired by cholesterol depletion (67). Similarly, cholesterol-lowering agents such as statins are known to inhibit HIV infection (68), probably by affecting membrane raft organization. It
remains that the exact role of slow and fast pools of cholesterol
(69) in raft stabilization should be taken into consideration for
future studies. In addition, Abs have been described to modulate
ceramide synthesis (7), together with enhanced Fas-mediated apoptosis and cell growth inhibition (7, 9). Ceramide and Abs liberate Ca2⫹ from the same intracellular pool (70). Agents able to
trigger ceramide synthesis can induce apoptosis, which is interesting for anticancer therapy (71), but they have also been described
to block HIV infection (72). These observed effects lead to the
following question: are the effects of lipid modulators and Abs
synergistic (70, 73)? If so, this could raise interesting avenues for
investigation. It is worth noting that lipid homeostasis inside rafts
is highly regulated (74). The tendency of cholesterol and ceramide
to avoid exposure to water might be a driving force for the association of proteins with rafts (74), such dynamics being mutually
regulated by the concentration of cholesterol and ceramide. For all
these reasons, the analysis of the lipid-protein rheostat in 37°Cextracted Brij 98 DRM, either upon treatment with biotechnological drugs or through a pathological process, could open new strategies for raft-based therapies.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Zap70 molecule (42), is physically disconnected from Zap70. Another possible means of modulation by 13B8.2 rIgG1 could involve
the other autophosphorylation sites Tyr292 and Tyr319 in the interdomain B of Zap70. The Tyr292 residue is a binding site for the Src
homology 2-like domain of the c-Cbl protooncogene and serves as
a negative regulator of T cell function. Phosphorylated Tyr292 interaction with c-Cbl, which is a component of the ubiquitination
machinery, may target phospho-Tyr292 Zap70 molecules for ubiquitination and degradation (57). Tyr319 residue is autophosphorylated in vitro and becomes phosphorylated in vivo upon TCR triggering (56), leading to positive regulation of TCR-mediated
signaling. The fact that CD4-specific rIgG1 13B8.2 induces Tyr292
phosphorylation of DRM-excluded Zap70, probably leading to increased kinase degradation, and inhibits Tyr319 phosphorylation,
as do the anti-CD4 YHB.46 (30), is one further enhancing element
for the switching-off of signaling pathways leading to impaired
immune responses or HIV infection. Decoupling of the Zap70
molecule from the raft machinery together with modulating its
phosphorylation are among the “missing links” to explain early
mechanisms of action of baculovirus-expressed anti-CD4 rIgG1
13B8.2. Are these mechanisms the only ones? Probably not, because 1) the disconnection of DRM-excluded Zap70 with adaptor
protein LAT and LIME has been observed (59); 2) anti-CD4 Abs
have been described to affect Zap70-induced phosphorylation of
the downstream adaptor proteins SLP-76, LAT, and Vav-1 (30 –
32), and we observed the rIg1 13B8.2-induced raft disconnection
of Zap70-dowstream targets SLP-76, Vav-1, and PLC␥1; and 3)
other adaptor proteins such as Nck and Wiskott-Aldrich syndrome
proteins (60) have been shown to localize with Zap70, linking the
TCR to actin polymerization.
An important finding from our confocal studies was that Zap70/
CD4 differential partitioning in Brij 98 DRM rapidly occurred
within 10 –30 s upon rIgG1 13B8.2 ligation, increased until 1 min
and persisted throughout the 1-h experiment. This observation
must be taken into consideration with regard to the dynamics of
signaling assemblies following TCR/CD3 ligation (61), which can
be potentially inhibited by 13B8.2 treatment. These clusters
quickly disassemble, reassemble, and change in composition as
specific proteins associate and dissociate. Zap70 is rapidly localized in patches (15 s) whose intensity reaches a steady state at 3
min; Zap70 remains associated with these laterally immobile clusters for 30 min (59). In contrast, adaptor signaling proteins such as
LAT, Grb2, Gads, and SLP-76 show similar initiation timing but
disappear rapidly, within 1–3 min, from these structures (61). This
phenomenon was also observed by Houtman et al. (62), who demonstrated that early Zap70 phosphorylation showed maximal effect
of 50% at 19 s following anti-CD3 T cell stimulation, occurred
maximally at 30 s, and persisted throughout the 120-s experiment.
Rapid (⬍5 min) and strong Ag-specific phosphorylation of Zap70
has been shown to be stable for 1 h and can be reduced by nondepleting anti-CD4 Abs KT6 and YTS177 (31). Ab-induced CD20
redistribution into Triton DRM occurs as early as 15 s, becomes
maximal at 15 min, and remains stable until the end of the 1-h
experiment (4). In comparison with these TCR or Ab dynamically
scheduled events, we can argue that 13B8.2-induced Zap70 exclusion from the raft platform interrupts this cross-talk early, that the
duration of the 13B8.2 effect strongly stabilizes cells in a
“pseudoanergized” form, and that Zap70 is a pertinent target because its TCR-induced dynamics were not found to be transient.
We demonstrated that the effect of rIgG1 13B8.2 on CD4/Zap70
differential raft partitioning is dependent on the Ab specificity toward the CDR3-like loop in the D1 domain of CD4. Numerous
unusual immunomodulatory or anti-HIV effects have already been
ascribed for ligands directed against this CDR3-like region in the
ANTI-CD4 13B8.2-INDUCED CD4/Zap70 RAFT PARTITIONING
The Journal of Immunology
419
Acknowledgments
We thank Annick Ozil, Nicole Bres, and Marylene Ozil for excellent technical assistance. We gratefully acknowledge Drs. D. Olive and C. Mawas
for providing the 13B8.2 mAb-producing cell line, Dr. F. Veas for supplying recombinant gp120, Dr. C. Gongora for providing the antiphosphotyrosine 4G10 Ab, and Dr. C. Germain for the recombinant anticarcinoembryonic Ag Ab. We are indebted to Dr. S. L. Salhi for
presubmission editorial assistance and M. Rigo for software assistance. We
are also indebted to the team from the Montpellier RIO Imaging platform
(P. Travo) for the confocal microscopy experiments.
Disclosures
21.
22.
23.
24.
The authors have no financial conflict of interest.
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ANTI-CD4 13B8.2-INDUCED CD4/Zap70 RAFT PARTITIONING