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IMMUNOBIOLOGY
SAP controls the cytolytic activity of CD8⫹ T cells against EBV-infected cells
Loı̈c Dupré, Grazia Andolfi, Stuart G. Tangye, Rita Clementi, Franco Locatelli, Maurizio Aricò, Alessandro Aiuti, and Maria-Grazia Roncarolo
The adaptor protein SAP regulates signaling through signaling lymphocytic activation molecule (SLAM)–family receptors
expressed on T and natural killer (NK)
cells. In patients affected by X-linked lymphoproliferative (XLP) disease, mutations
in the SH2D1A gene result in defective
lytic activity. However, the mechanism by
which SAP controls cytotoxic activity remains unclear. T-cell–receptor (TCR) activation of CD8ⴙ cytotoxic T cells (CTLs)
results in down-regulation of SAP, suggesting that this protein is involved in
early activation events. Here, we show
that SAP-deficient CTLs from patients
with XLP and hemophagocytic lymphohistiocytosis (HLH) display a specific lytic
defect against autologous and allogeneic
Epstein-Barr virus (EBV)–positive B cells.
This defect is associated with the defective polarization of 2B4, perforin, and lipid
rafts at the contact area of CTLs with
EBV-positive targets. Blockade of 2B4 in
normal CTLs reproduces the defects in
lysis and polarization observed in SAPdeficient CTLs. Expression and regulation of the SLAM-family receptors SLAM,
CD84, and 2B4, as well as the lytic effec-
tors perforin and granzyme-B are normal
in SAP-deficient CTLs. In addition, TCR
stimulation leads to normal proliferation
and production of interleukin 2 (IL-2),
IL-4, and interferon-␥ (IFN-␥). These results demonstrate that the SAP/2B4 pathway plays a key role in CTL lytic activity
against EBV-positive targets by promoting the polarization of the lytic machinery.
(Blood. 2005;105:4383-4389)
© 2005 by The American Society of Hematology
Introduction
X-linked lymphoproliferative (XLP) disease is a severe immunodeficiency characterized by variable clinical phenotypes, including
fatal infectious mononucleosis, malignant B-cell lymphoma, and
progressive dysgammaglobulinemia.1 The gene responsible for
XLP (SH2D1A) encodes for SAP (SLAM [signaling lymphocyteactivation molecule]–associated protein), or SH2D1A, a natural
killer (NK) and T-cell–specific signaling adaptor protein.2-4 Mutations in the SH2D1A gene have been detected in patients with
hemophagocytic lymphohistiocytosis (HLH)5 and common variable immunodeficiency syndrome,6,7 thereby expanding the range
of clinical phenotypes associated with this genetic defect. These
heterogeneous clinical manifestations might be related to viral
infections, which appear to act as secondary factors triggering the
severe forms of SAP-deficiency syndrome. In the absence of SAP,
dysregulation of T/B-cell interactions and NK-cell functions has
been proposed to result in a variable ability to control Epstein-Barr
virus (EBV) infection, leading to either fatal infectious mononucleosis, or infectious mononucleosis with progression toward malignant B-cell lymphoma or acquired agammaglobulinemia.8 However, in some patients with XLP, these manifestations can also
occur in the absence of EBV infection.9
SAP is a protein of 128 residues with a single Src homology 2
(SH2) domain binding to a consensus T.I/V.(p)Y.x.x.V/I motif on
the cytoplasmic tail of surface molecules of the SLAM family,
including SLAM, 2B4, CD84, Ly-9, and NTB-A.2,10-13 These
molecules form homotypic and heterotypic receptor-ligand pairs
during T cell/antigen-presenting cell or NK cell/target cell contacts.
SLAM is expressed on activated and memory T cells, in which it
can skew cytokine production toward a T helper 1 (Th1) profile14
and can act as a costimulatory molecule for cytotoxic activity.15
SAP appears to be a natural inhibitor of the interaction of
SLAM-family members with SH2-containing signal molecules
such as the SH2-containing phosphatase 2 (SHP-2) phosphatase.2
In T cells, SAP recruits FynT, which is required for SLAM
phosphorylation. Activation of the SLAM/SAP pathway controls
the phosphorylation of dok1, dok2, and SH2-containing inositol
phosphatase, thereby playing a potential role in the control of
interferon-␥ (IFN-␥) production.16,17 Furthermore, SAP appears to
participate in the adhesion of T cells to target cells, as well as in
T-cell receptor (TCR)–mediated activation.18,19 In SAP-deficient
murine models,20,21 infection with lymphocytic-chorio-meningitis
virus is associated with higher mortality related to increased T-cell
activation and IFN-␥ production, as well as to a reduction in the
production of interleukin 4 (IL-4) and the generation of immunoglobulin-secreting cells. However, in this model, T- and NK-cell
cytotoxic activities have not been investigated.
A number of studies in patients with XLP indicate that 2B4
stimulation on NK cells induces an abnormal dominant abrogation
of NK-cell lytic activity. This defect is also present when EBVinfected cells are used as targets.22-24 Expression of CD48, the
natural ligand of 2B4, is strongly increased on B cells upon EBV
infection, while expression of major histocompatibility complex
From the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET),
Milan, Italy; the Centenary Institute of Cancer Medicine and Cell Biology,
Newtown, Australia; the Pediatric Hematology/Oncology, Istituto di Ricovero e
Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo, Pavia, Italy; the
Children’s Hospital G. Di Cristina, Palermo, Italy; and the Vita-Salute San
Raffaele University, Milan, Italy.
Supported by a core grant from the Italian Telethon Foundation (M.-G.R.) and
the New South Wales Cancer Council (S.G.T.).
Submitted August 24, 2004; accepted December 30, 2004. Prepublished
online as Blood First Edition Paper, January 27, 2005; DOI 10.1182/blood2004-08-3269.
BLOOD, 1 JUNE 2005 䡠 VOLUME 105, NUMBER 11
Reprints: Maria-Grazia Roncarolo, San Raffaele Telethon Institute for Gene
Therapy, via Olgettina 58, 20132 Milan, Italy; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2005 by The American Society of Hematology
4383
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4384
DUPRÉ et al
molecules is down-regulated.25 Thus, EBV infection may lead to a
specific triggering of 2B4, thereby inducing a progressive attrition
of SAP-defective NK-cell function. Initial studies on the function
of T cells in patients with XLP gave controversial results that could
be related to the different clinical stages and phenotypes of the
patients studied.26,27 A recent report showed reduced IFN-␥ production and lytic activity by EBV-specific cytotoxic T lymphocytes
(CTLs) in 3 patients with XLP.28 However, the mechanism
underlying this defective lytic activity of SAP-deficient CD8⫹ T
cells was not identified. In addition, the respective regulation of
expression of the SLAM-family receptors and of SAP in EBVspecific CTLs was not studied. In this study, we found that the
defective lytic activity of SAP-deficient CD8⫹ T cells is specific to
EBV-infected target cells. SLAM-family receptors are expressed
normally, and perforin and granzyme-B are also produced. However, SAP-deficient CD8⫹ T cells fail to polarize 2B4, perforin, and
the lipid raft marker GM1 at the contact with EBV-positive target
cells. Our results identify the 2B4/SAP pathway as an important
regulator of the assembly of the lytic synapse that forms between
CD8⫹ CTLs and EBV-positive B-cell targets.
BLOOD, 1 JUNE 2005 䡠 VOLUME 105, NUMBER 11
formula: 100 ⫻ (cpm experimental release ⫺ cpm spontaneous release) /
(cpm maximum release ⫺ cpm spontaneous release).
Cell proliferation and cytokine production
Resting T cells were plated at 1 ⫻ 105 cells/well in 96-well flat-bottom
plates precoated with the indicated dose of anti-CD3 mAbs (OKT3;
Janssen-Cilag, Milan, Italy). After 72 hours, cells were labeled with
3H-thymidine for 18 hours. Thymidine incorporation was measured in
triplicates by liquid scintillation counting. IL-2, IL-4, and IFN-␥ production
was measured by intracytoplasmic fluorescence-activated cell sorting
(FACS) analysis of cells stimulated for 3 hours with either 1 ␮g/mL
immobilized anti-CD3 mAbs plus 10 ␮g/mL anti-CD28 mAbs (BD
Biosciences Pharmingen, San Diego, CA) or with 10 ng/mL TPA (12-Otetradecanoylphorbol acetate) plus 500 ng/mL ionomycin. Cells were
treated with 10 ␮g/mL brefeldin A and were then incubated additionally for
3 hours. Cells were fixed in 2% formaldehyde, permeabilized with 0.5%
Saponin, and subsequently stained with phycoerythrin (PE)–labeled anti–
IL-2, PE-labeled anti–IL-4, and fluorescein isothiocyanate (FITC)–labeled
anti–IFN-␥ (BD Biosciences Pharmingen). After washing, cells were
analyzed by using a FACScan flow cytometer (BD Biosciences, San Diego,
CA) and CellQuest software (BD Biosciences).
FACS analysis of CTL activation markers
Patients, materials, and methods
Patients and cells
Patient 1 was diagnosed as HLH and previously described.5 His SH2D1A
gene sequence carries a single nucleotide mutation in exon 3 resulting in a
stop codon (Tyr76⬘STOP). Patients 2 and 3 were diagnosed as XLP and
have been described previously.12 Patient 2 carries a single nucleotide
mutation in exon 2, resulting in a Tyr54Cys amino acid substitution. Patient
3 carries a single nucleotide mutation in exon 3, resulting in a Phe87Ser
amino acid substitution. Blood samples from patients and healthy donors
were obtained following standard ethical procedures (Helsinki protocol)
and with the approval of the concerned internal review boards.
T-cell lines
T cells from SAP-deficient patients and healthy control donors were derived
from peripheral blood mononuclear cells (PBMCs) isolated with Lymphoprep (Nycomed Pharma AS, Oslo, Norway). For the generation of
allogeneic T-cell lines, PBMCs were stimulated every 2 weeks at the
concentration of 2 ⫻ 105 cells/mL with a feeder cell mixture which
comprises irradiated allogeneic PBMCs (1 ⫻ 106/mL), irradiated EBVtransformed JY cells (1 ⫻ 105/mL), phytohemagglutinin (1 ␮g/mL), IL-2
(100 IU/mL), and IL-7 (10 ng/mL). For the generation of EBV-specific
T-cell lines, PBMCs were stimulated every 2 weeks at the concentration of
5 ⫻ 105 cells/mL with 1 ⫻ 105/mL irradiated autologous EBV-transformed
B-cell lines (B-EBV), IL-2 (100 IU/mL), and IL-7 (10 ng/mL). T-cell lines
were cultured in Yssel medium (Dyaclone, Besançon, France), 10% fetal
calf serum (or 5% human serum for the EBV-specific T-cell lines), and
penicillin and streptomycin. CD8⫹ T cells were purified after 4 rounds of
stimulation by depleting CD4⫹ T cells with anti-CD4 antibody (Abs)–
coated magnetic beads (Dynal AS, Oslo, Norway).
Cytotoxic assays
Cytotoxic activity was measured in a standard 51Chromium (51Cr) release
assay as described previously.29 Briefly, 1000 51Cr-labeled target cells,
including JY, K562, and autologous B-EBV, were incubated for 4 hours
with effector CD8⫹ T cells at the indicated effector-target ratios at 37°C in
5% CO2. Blocking anti-2B4 monoclonal antibodies (mAbs; Clone c1.7;
Immunotech, Marseille, France) were added at a concentration of 5 ␮g/mL
prior to adding the labeled target cells on the effector cells. The radioactivity liberated in the supernatant by the lysed targets was measured with a
␥-counter. Percentage of specific lysis was calculated according to the
Resting CD8⫹ T cells were incubated for 24 hours at 1 ⫻ 106/mL in the
presence of either 100 ng/mL IL-15 (R&D Systems, Abingdon, United
Kingdom) or 1 ␮g/mL immobilized anti-CD3 mAbs. T cells were washed
and resuspended in phosphate-buffered saline (PBS) containing 0.3%
bovine serum albumin (BSA) and 0.1% Na-azide before incubation for 15
minutes at 4°C with one of the following mAbs: PE-conjugated anti-CD25
mAbs, FITC-conjugated anti-CD69 mAbs, PE-conjugated mouse antiCD84 mAbs (BD Biosciences Pharmingen), phycoerythrin-cyanin 5–conjugated mouse anti-2B4 mAbs (Immunotech), and purified anti-SLAM mAbs
(gift from Dr Aversa, DNAX Research Institute, Palo Alto, CA). For
anti-SLAM staining, in a second step cells were incubated for 15 minutes at
4°C with 5 ␮g/mL PE-conjugated goat anti–mouse immunoglobulin G
(IgG) Abs (Southern Biotechnology Associates, Birmingham, AL). For
staining of the intracellular proteins perforin and granzyme-B, T cells were
fixed and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences
Pharmingen), blocked with 5% normal rabbit serum, and incubated with
PE-conjugated anti–perforin mAbs or purified anti–granzyme-B mAbs (BD
Biosciences Pharmingen) for 20 minutes at 4°C. For anti–granzyme-B
staining, in a second step cells were incubated for 15 minutes at 4°C with
PE-conjugated goat anti–mouse IgG Abs. Stained cells were washed and
analyzed by FACS as described in “Cell proliferation and cytokine
production.”
Immunofluorescence
Resting T cells were plated at 1 ⫻ 106/well on 96-well U-bottom plates, in a
final volume of 100 ␮L. Target cells (B-EBV, JY, or K562) were added at
the concentration of 2 ⫻ 106/100 ␮L. Plates were centrifuged for 10
seconds at 1500 rpm (300 ⫻ g) and incubated for 20 minutes at 37°C. Cell
conjugates were transferred onto poly-L-lysine–coated coverslips for 20
minutes at 37°C and subsequently fixed with 4% paraformaldehyde for 30
minutes at room temperature. Staining for GM1 and perforin was performed by incubating the cells in PBS, 0.3% BSA, 0.3% saponin with 8
␮g/mL FITC-conjugated Cholera toxin subunit-B (Sigma, St Louis, MO),
and PE-conjugated anti-perforin mAbs (used at the dilution recommended by the
manufacturer; BD Biosciences Pharmingen). Staining for 2B4 was performed by
incubating the cells in PBS, 0.3% BSA, with 10 ␮g/mL anti-2B4 mAbs
(Immunotech), and subsequently with Alexa 488–conjugated goat anti–mouse
secondary Abs (Molecular Probes, Eugene, OR). Each staining step was
performed at room temperature for 45 minutes. After washing, coverslips were
mounted with Fluoromount-G (Southern Biotechnology Associates) and analyzed on an Olympus Provis AX70 microscope using a 100 ⫻ immersion
objective lens (numerical aperture 1.30; Olympus, Melville, NY). Images were
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BLOOD, 1 JUNE 2005 䡠 VOLUME 105, NUMBER 11
SAP CONTROLS CTL ACTIVITY AGAINST EBV
4385
Defective lytic activity of SAP-deficient CD8ⴙ T cells against
EBV-positive targets
Figure 1. Regulation of SAP expression in CD8ⴙ T cells. Western blot analysis of
SAP expression in CD8⫹ T cells from healthy donors (ND1, ND2, and ND3), and
patients with HLH (SAP1) and XLP (SAP2 and SAP3) at a resting state and at 24 or
72 hours after activation with 10 ␮g/mL immobilized anti-CD3 mAbs.
acquired with a Zeiss Axiocam camera using Zeiss Axiovision 3.1 software (Carl
Zeiss, Jena, Germany).
Western blot protein analysis
Cell lysates were prepared from 1 ⫻ 106 PBS-washed cells in 20 ␮L lysis
buffer (20 mM Tris (tris(hydroxymethyl)aminomethane) pH 7.5, 150 mM
NaCl, 1% Nonidet P40 (Octylphenolpoly[ethyleneglycolether]n), 2 mM
EDTA (ethylenediaminetetraacetic acid)) supplemented with 100 ␮g/mL
phenylmethlsulfonyl fluoride and a complete set of protease inhibitors
(Roche Molecular Biomedicals, Mannheim, Germany). After 30 minutes on
ice, lysates were centrifuged, and supernatant was resuspended in denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) sample buffer. An aliquot of each lysate was used for total protein
concentration determination with the bicinchoninic acid protein assay
(Pierce, Rockford, IL) to normalize each sample. Following SDS-PAGE
resolution, proteins were transferred onto nitrocellulose membranes, and
correct loading and transfer were checked by Ponceau staining. Incubation
with anti–SAP rabbit polyclonal antibody (kindly provided by Dr Nichols,
Children’s Hospital of Philadelphia, PA) diluted 1:500 in 5% milk in
Tris-buffered saline–Tween 20 (0.05%) was performed. Horseradish peroxidase–coupled anti–rabbit Abs (Dako A/S, Glostrup, Denmark) were used at
the dilution of 1:2000 as secondary Abs, and detection was performed with
enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Little
Chalfont, United Kingdom) detection system.
The lytic activity of SAP-deficient T cells was first investigated by using
a system in which EBV antigens were presented by autologous B cells.
To generate antigen-specific CTLs, CD8⫹ T cells from the patient with
HLH (SAP1) were stimulated repeatedly with autologous EBVtransformed B cells. EBV-specific CD8⫹ T cells derived from patient
SAP1 displayed a reduced lytic activity against autologous EBVpositive B-cell targets, when compared with healthy EBV-specific
CD8⫹ T cells generated in parallel (Figure 2A). Next, we investigated
the lytic activity of SAP-deficient CTLs against allogeneic EBVinfected B-cell targets. To this aim, CD8⫹ T cells from the 3 SAPdeficient patients were repeatedly stimulated with EBV-positive JY cells
and then tested for their lytic activity against these targets. SAP-deficient
CTLs had a dramatically reduced ability to lyse JY target cells. At an
effector-target ratio of 90:1, SAP-deficient CD8⫹ T cells had a lytic
activity of 8%, whereas SAP-positive control CD8⫹ T cells had a lytic
activity of 46% (Figure 2B). The lytic activity of normal CTLs against
JY cells was mediated through 2B4, since preincubation with anti-2B4
mAbs resulted in a significant inhibition, with percentages of lysis
comparable to those of SAP-deficient CTLs (Figure 2C). In contrast,
when the K562 cell line (EBV negative and CD48⫺) was used as target,
the lytic activity of the SAP-deficient CD8⫹ T cells was not impaired.
Indeed, SAP-deficient CTLs from the 3 patients were able to lyse K562
cells at even higher levels than control CD8⫹ T cells (Figure 2D). These
results indicate that the lytic capacity of SAP-deficient CD8⫹ T cells is
not intrinsically impaired, but that activation through 2B4, which is
required for EBV-specific T-cell cytotoxic activity, is defective.
Results
SAP deficiency in CD8ⴙ T cells from patients with HLH and XLP
Untransformed CD8⫹ T-cell lines were established from 3 healthy
donors (ND1, ND2, and ND3), 1 patient with HLH (SAP1), and 2
patients with XLP (SAP2 and SAP3) with SH2D1A gene point
mutations, by repeated stimulation with allo-PBMCs and the
EBV-positive cell line JY. CD8⫹ T cells from the healthy donors
expressed high levels of the SAP protein at the resting stage (9-10
days after stimulation), whereas SAP expression was strongly
down-regulated 24 and 72 hours after TCR stimulation with
anti-CD3 mAbs (Figure 1). Resting CD8⫹ T cells from patients
with HLH and XLP failed to show detectable expression of the
SAP protein (Figure 1). Interestingly, the SAP mRNA levels
detected by reverse transcription–polymerase chain reaction in
normal cells, before and after TCR stimulation, were comparable to
those from SAP-deficient patients were comparable (data not
shown). These data demonstrate that in untransformed human
CD8⫹ T cells, the expression of SAP protein is tightly regulated
during activation and suggest that SAP plays a role relatively early
during T-cell activation.
Figure 2. Lytic activity of SAP-deficient CD8ⴙ T cells. (A) Lytic activity of
EBV-specific CD8⫹ T-cell lines from a SAP-deficient HLH patient (SAP1) and from a
healthy control donor. The targets are autologous EBV-transformed B-cell lines used
at the indicated effector-target ratio. (B) Mean (⫾ SD) of the lytic activity of allospecific
CD8⫹ T-cell lines from 3 SAP-deficient patients (SAP1, SAP2, and SAP3) and from 3
healthy donors (ND1, ND2, and ND3) against the EBV-transformed B-cell line JY.
(A-B) f indicates ND; 䡺, SAP deficient. (C) Mean (⫾ SD) of the lytic activity of
allospecific CD8⫹ T-cell lines from 3 healthy donors (ND1, ND2, and ND3) against JY
cells in the presence of either blocking anti-2B4 mAbs (o) or isotype control Abs
(⫹IgG1; f). (D) Mean (⫾ SD) of the lysis of allospecific CD8⫹ T-cell lines from 3
SAP-deficient patients (SAP1, SAP2, and SAP3) and from 3 healthy donors (ND1,
ND2, and ND3) against the CD48⫺ cell line K562. f indicates ND; 䡺, SAP deficient.
One representative experiment of 5 performed is shown. Each mean (⫾ SD) was
obtained from 6 data points (3 cell lines studied in duplicate), and statistical analysis
was performed using an unpaired t test. Significant differences of lysis between SAP
and control T-cell lines are indicated by asterisks (*P ⬍ .05; **P ⬍ .01).
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4386
DUPRÉ et al
BLOOD, 1 JUNE 2005 䡠 VOLUME 105, NUMBER 11
Defective polarization of perforin, GM1, and 2B4
in SAP-deficient CD8ⴙ T cells
We next investigated the ability of SAP-deficient CTLs to polarize
the lytic machinery toward the site of contact with EBV-infected
target cells. The lytic activity of both NK cells and CTLs has been
shown to depend on the assembly of a specialized immunologic
synapse, which occurs through reorganization of lipid rafts,
surface receptors, signaling molecules, cytoskeleton, and lytic
granules. By immunofluorescence analysis of control and SAPdeficient CD8⫹ T cells conjugated to EBV-infected targets, we
investigated the orientation of both perforin and ganglioside
GM1 as markers for lytic granules and lipid rafts, respectively.
Similarly to control CTLs, SAP-deficient CTLs were able to
form conjugates with JY cells and were positive for perforin and
GM1 (Figure 3A). In control CTLs, polarization of both GM1
and perforin at the contact area with JY cells was observed in
36% of the cells. In contrast, only 9% of the SAP-deficient
CTLs, which formed conjugates with JY cells, polarized GM1,
and perforin at the site of contact (Figure 3B). Importantly,
incubation of control CTLs with blocking anti-2B4 mAbs
resulted in a significative decrease in the percentage of cells
Figure 4. Distribution of 2B4 in normal and SAP-deficient CD8ⴙ T cells. (A) 2B4
distribution in normal CD8⫹ CTLs (T ) either alone or at the contact with EBV⫹/CD48⫹
JY B cells or EBV⫺/CD48⫺K562 cells. Representative cells are shown as bright field
(i-iv) and 2B4 staining (v-viii). The white arrows indicate 2B4 clustering at the cell
contact area. (B) 2B4 distribution in SAP-deficient CD8⫹ T cells either alone or at the
contact with EBV⫹/CD48⫹ JY B cells. Representative cells are shown as bright field
(i-ii) and 2B4 staining (iii-iv). (C) Quantitative analysis of the percentage of CTLs
(normal or SAP deficient) with clustered 2B4 at the cell contact area. f indicates
normal CTL ⫹ JY; u, normal CTL ⫹ K562; and 䡺, SAP-deficient CTL ⫹ JY cells.
Mean percentages (⫾ SDs) of 3 experiments counting T cells forming clusters with
single target cells (100 conjugates were counted). Statistical analysis was performed
using an unpaired t test, and P values corresponding to the comparison of 1 group
with the other are indicated.
Figure 3. Distribution of perforin and GM1 in SAP-deficient CD8ⴙ T cells. (A)
GM1 and perforin distribution in normal and SAP-deficient CD8⫹ T cells (T ) forming
conjugates with EBV-positive B-cell line JY (B ). The effects of blocking anti-2B4
mAbs on normal CD8⫹ T cells are also shown. One representative conjugate is
shown in parallel as bright field (i-iii), GM1 staining (iv-vi), and perforin staining (vii-ix).
(B) Quantitative analysis of GM1 and perforin coclustering at the area of contact with
B-cell targets in T cells from 3 SAP-deficient patients and T cells from 3 healthy
donors, either untreated or treated with blocking anti-2B4 mAbs. Cells were
considered positive for coclustered GM1 and perforin if the staining was centered at
the site of contact with the B cell and occupied less than one third of the cell surface.
Data are represented as mean percentages (⫾ SDs) of 3 experiments counting T
cells forming clusters with a single JY B-cell target (for each cell line a total of 250-300
cells was counted). Statistical analysis was performed using an unpaired t test, and P
values corresponding to the comparison of 1 group with the other are indicated.
with polarized GM1 and perforin (14%). A defect in GM1 and
perforin polarization was also observed when autologous EBVlymphoblastoid cells were used as targets of EBV-specific CTLs
from patient SAP1, whereas normal polarization occurred when
K562 cells were used as targets (data not shown). Our results
indicate that in the absence of stimulation through 2B4 or SAP,
the reduced lytic activity of CTLs against EBV-positive cells is
associated with a defect in the orientation of both lipid rafts and
lytic granules at the effector-target contact area.
We next investigated the role of SAP in the localization of
2B4 during the lytic process. In resting normal CD8⫹ T cells,
2B4 was distributed all over the cell surface in small discrete
clusters (Figure 4Av). The absence of SAP in CD8⫹ T cells from
the patients had no effect on the distribution of 2B4 at the resting
stage (Figure 4Biii). Upon contact with JY target cells, normal
CD8⫹ T cells redistributed 2B4 to the site of effector-target
contact area, either as a central cluster (Figure 4Avi) or as a
continuous pattern covering the entire length of the contact area
(Figure 4Avii). The percentage of CD8⫹ T cells with 2B4
clustering (Figure 4C) was very similar to the percentage of
CD8⫹ T cells with GM1/perforin polarization. In contrast,
SAP-deficient CD8⫹ T cells failed to cluster 2B4 at the contact
area (Figure 4Bii,iv and 4C), indicating that SAP is required for
the redistribution of 2B4 to the lytic immunologic synapse.
Importantly, no polarization of 2B4 could be detected when
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BLOOD, 1 JUNE 2005 䡠 VOLUME 105, NUMBER 11
SAP CONTROLS CTL ACTIVITY AGAINST EBV
4387
K562 cells were used as targets (Figure 4Aviii and 4C),
indicating that 2B4 clustering requires interaction with the
CD48 ligand. Our results indicate that the 2B4/SAP pathway is
crucial for the assembly of the lytic immunologic synapse at the
contact of CD8⫹ T cells with EBV-infected target cells.
SAP-deficient CD8ⴙ T cells express normal levels of activation
markers and lytic molecules
In addition to a defect in lytic immunologic synapse assembly, the
defective lytic activity of CD8⫹ T cells from patients with XLP and
HLH could also be the result of an inappropriate activation.
SAP-deficient CTLs showed normal intracytoplasmic expression
of the 2 major lytic mediators, perforin and granzyme-B. The levels
of perforin and granzyme-B, before and after activation with
anti-CD3 mAbs or stimulation with IL-15, were comparable both
in SAP-deficient and control CTLs (Figure 5A). Furthermore, the
expression of SLAM, CD84, and 2B4, which are the bestcharacterized SAP-binding receptors, in resting and activated
CTLs from SAP-deficient and control CTLs was similar. In the
resting stage, CTLs expressed very low levels of SLAM, whereas
most of the cells showed to be positive for CD84 and 2B4 (Figure
5B). However, TCR activation induced a rapid up-regulation of
SLAM, with the majority of the cells becoming positive. Similarly,
the levels of CD84 and 2B4 increased, as measured by mean
fluorescence intensity, following TCR stimulation. Interestingly,
stimulation with IL-15 also led to up-regulation of SLAM, but it
had no effect on the expression of CD84 and 2B4 (Figure 5B). The
activation markers CD25, CD56, and CD69 were expressed at
similar levels in SAP-deficient and control CTLs, prior to and after
stimulation through the TCR or with IL-15 (Figure 5C). Therefore,
SAP-deficient CD8⫹ T cells acquire the typical phenotype of activated
CTLs upon stimulation either through the TCR or with IL-15.
Figure 6. Proliferation and cytokine production of SAP-deficient CD8ⴙ T cells.
(A) Proliferation of CD8⫹ T-cell lines from healthy donors (mean ⫾ SD of ND1, ND2,
and ND3; f) and SAP-deficient patients (mean ⫾ SD of SAP1, SAP2, and SAP3; 䡺)
after stimulation with the indicated doses of immobilized anti-CD3 mAbs. Proliferation
is expressed as cpm values corresponding to 3H-thymidine uptake after a 72-hour
stimulation. (B) Intracytoplasmic staining for cytokine production in T cells of healthy
donors and SAP-deficient patients 6 hours after stimulation with immobilized
anti-CD3 mAbs plus anti-CD28 mAbs or TPA/ionomycin. Numbers in dot plot
quadrants refer to percentages of T cells positive for the indicated cytokines. One
representative experiment of 3 performed is shown.
Normal proliferation and cytokine production in SAP-deficient
CD8ⴙ T cells
We then investigated whether SAP-deficient CD8⫹ T cells had a
general defect in TCR-mediated activation, leading not only to
impaired lytic activity but also to defects in proliferation and
cytokine production. SAP-deficient CD8⫹ T cells from patients
SAP1, SAP2, and SAP3 proliferated normally after TCR stimulation with immobilized anti-CD3 mAbs (Figure 6A). The production of cytokines, including IL-2, IL-4, and IFN-␥, was normal in
CD8⫹ T cells from patients with SAP1 (HLH) and SAP3 (XLP),
both after stimulation with anti-CD3/anti-CD28 mAbs or with
TPA/ionomycin (Figure 6B). The normal cytokine production by
SAP-deficient CD8⫹ T cells was confirmed by enzyme-linked
immunosorbent assay (data not shown). These data indicate that,
although the cytotoxic activity against B-cell targets presenting
EBV antigens was impaired in the CTLs of patients, signaling
through the TCR, which leads to proliferation and cytokine
production, was preserved.
Figure 5. Phenotype of SAP-deficient CTLs. Phenotype of CD8⫹ T cells from
SAP-deficient patients (SAP; 䡺) and healthy donors (ND; f) in a resting state or after
activation with 1 ␮g/mL anti-CD3 mAbs or stimulation with 100 ng/mL IL-15 (mean ⫾
SD of 3 SAP-deficient patients and 3 healthy donors is shown). (A) Mean fluorescence intensity of the intracytoplasmic staining of the lytic molecules perforin and
granzyme-B. (B) Percentage of CD8⫹ T cells staining positive for the CD2superfamily receptors SLAM, 2B4, and CD84. (C) Percentage of CD8⫹ T cells
staining positive for the T-cell activation markers CD25, CD69, and CD56. One
representative experiment of 3 performed is shown.
Discussion
The clinical onset of XLP is usually triggered by EBV infection.
The defective lytic activity of NK cells has been suggested to
account for the inappropriate response to EBV-infected B cells and
for the development of the pathologic phenotypes of XLP.23,24 Our
study, together with the recent findings by Sharifi et al28 in patients
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4388
DUPRÉ et al
with XLP, demonstrate that the cytotoxic activity of SAP-deficient
CD8⫹ T cells from patients with XLP and HLH toward EBVinfected B-cell targets is also impaired. Therefore, the clinical
manifestations of SAP-deficiency syndromes may result from a
synergistic defect in T- and NK-cell–mediated lysis of EBVinfected B cells. In the present study, we show that the absence of
SAP (in CTLs from patients) or the blockade of 2B4 (in normal
CTLs) results in a specific defective CTL activity toward EBV-infected
targets. This lytic defect is associated with a defect in polarization of
2B4, perforin, and lipid rafts, indicating that signaling through 2B4 and
SAP controls and promotes the polarization of the lytic machinery of
CTLs at the site of contact with EBV-infected target cells.
Although CD8⫹ T cells from patients with SH2D1A gene
mutations have a defective lytic activity against EBV-infected
cells, they have the phenotype of effector CTLs. The activation
markers CD25, CD69, CD56 are normally up-regulated in SAPdeficient CD8⫹ T cells following activation through the TCR or with
IL-15. Furthermore, the SAP-interacting receptors SLAM, 2B4, and
CD84, which costimulate lytic activity,15,30 are normally expressed in
resting or activated CD8⫹ T cells from SAP-deficient patients. These
results show that SAP deficiency does not impair the expression of these
receptors. In addition, the levels of expression and regulation of
granzyme-B and perforin are also normal in SAP-deficient CD8⫹ T
cells, which can form conjugates with EBV-infected target cells,
indicating that these cells are equipped to mediate lysis.
However, the polarization of the lytic mediator perforin toward
the cell-cell contact area is significantly reduced in CD8⫹ T cells
from patients with XLP and HLH, and this can be responsible for
the lytic defect. Indeed, effective lysis requires the rapid polarization of perforin-containing lytic granules to the center of the lytic
immunologic synapse that forms at the CTL-target cell contact.31-33
The polarization of the lipid raft marker GM1, which has been
shown to cluster at the immunologic synapse,34 is also reduced in
CTLs lacking SAP. In conclusion, the absence of SAP impairs the
polarization of both lytic granules and lipid rafts at the contact of
the CTL with the target cell, suggesting a general defect in the
assembly of the lytic immunologic synapse. This defect is restricted to EBV-positive CD48⫹ target cells since both the polarization of perforin and GM1 and the lytic activity against the
EBV-negative K562 targets are not reduced in SAP-deficient CTLs.
SAP might be specifically required for the activation of CTLs
against EBV because of the sustained stimulation through 2B4
induced by EBV-infected B cells. Indeed, expression of CD48, the
natural ligand of 2B4, is strongly increased on B cells upon EBV
infection.25 In this context, the absence of SAP might be much
more critical than during infection by other intracellular pathogens.
Our data show that expression of both 2B4 and SAP are associated
to a high lytic activity of normal CTLs. In contrast, SLAM is not
expressed in active CTLs and requires stimulation with anti-CD3
mAbs or IL-15 to be up-regulated. Given the rapid down-regulation
of SAP expression observed after TCR activation, it is likely that in
human CTLs, SAP preferentially signals through 2B4, rather than
through SLAM, during the first minutes of stimulation needed for
the activation of the lytic machinery. In agreement with this
hypothesis, experiments with blocking anti-2B4 mAbs demonstrated that the lytic activity of normal CTLs against JY cells, as
well as the polarization of perforin, is mediated through 2B4.
Interestingly, there is evidence that 2B4 is involved in the assembly
of the immunologic synapses that form between NK cells and their
BLOOD, 1 JUNE 2005 䡠 VOLUME 105, NUMBER 11
targets.35,36 Recent studies show that 2B4 and SAP are rapidly
recruited to the central part of the NK immunologic synapse that
forms at the contact with CD48⫹ target cells.37,38 In addition, it has
been proposed that killer cell inhibitory receptors (KIRs) negatively control NK immunologic synapse assembly and lytic activity
by blocking recruitment of 2B4 into lipid rafts and by blocking
clustering of lipid rafts at the synapse in a SHP-1– and SHP-2–
dependent manner.34,39 Since SAP has the properties to compete
with SHP-2 and to bind to 2B4, it is possible that SAP regulates the
activity of both activatory (2B4) and inhibitory (KIR) receptors
controlling the assembly of the lytic synapse. Similarly to the
localization of 2B4 to the NK immunologic synapse, we found that
2B4 is recruited to the immunologic synapse formed between
normal CD8⫹ CTLs and EBV-positive targets. In contrast, in the
absence of SAP, 2B4 is not recruited to the site of the immunologic
synapse and is confined to subtle patches distributed over the
surface of the CTLs. This demonstrates that SAP is required for the
recruitment of 2B4 to the site of the immunologic synapse. In
addition, no 2B4 clustering was observed in normal CTLs forming
conjugates with CD48⫺ targets (K562), suggesting that the SAPdependent 2B4 relocalization is restricted to EBV-specific CTLs.
Together, our results indicate that in SAP-deficient CTLs, defective
relocalization of 2B4 during encounter with a CD48⫹ target cell
(EBV-infected) results in failure to organize the lytic synapse.
SAP expression is tightly regulated during TCR-driven activation of human untransformed CD8⫹ T cells. Indeed, after 24- and
72-hour stimulation, the levels of SAP protein are strongly
down-regulated, confirming data obtained in murine T cells.40
Since we show that signaling through 2B4 and SAP is crucial for
the assembly of the lytic immunologic synapse and for the lytic
activity of EBV-specific CTLs, it is possible that the downregulation of SAP expression decreases CTL activation after the
killing of target cells has occurred. Our data point to a posttranscriptional regulation of the SAP protein since SAP mRNA is not
down-regulated following TCR stimulation (data not shown). In
addition, patients’ T cells express normal levels of point-mutated
SAP mRNA, but no protein is detected. Accordingly, we found that
retroviral vector–mediated SH2D1A gene transfer into T cells of
SAP-deficient patients results in very limited levels of SAP protein
expression, despite the presence of SAP mRNA. This limited
expression of the SAP protein after gene transfer was not sufficient
to restore the ability of patients’ T cells to lyse target cells and to
polarize perforin and GM1 (data not shown).
Besides acting as costimulatory molecules to enhance cytotoxic
activity, SLAM-family receptors, such as SLAM and CD84, are
involved in the pathway regulating IFN-␥ production.11,14,41 Sharifi
et al28 demonstrated that SAP-deficient EBV-specific T-cell lines
had a reduced frequency of IFN-␥–producing cells upon TCR or
TCR/2B4 stimulation in parallel to having a defect in the lysis of
autologous EBV-positive target cells. In addition, Sanzone et al18
reported a role of SAP for TCR signaling, proliferation, IL-2
production, and adhesion. However, no defects in proliferation,
cytokine production, and CD25 up-regulation were observed in the
SAP-deficient T-cell lines described here. This discrepancy could
be due to differences in the clinical stage and history of infection of
the patients investigated.
Together, our results indicate that SAP-deficient CD8⫹ T cells
from patients with XLP and HLH have defects in the regulation of
cytotoxic activity against EBV-positive targets. Although the
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 1 JUNE 2005 䡠 VOLUME 105, NUMBER 11
SAP CONTROLS CTL ACTIVITY AGAINST EBV
phenotype of these CD8⫹ T cells is normal, they have a reduced
ability to polarize key components of the lytic immunologic
synapse at the contact with EBV-positive target cells. Together with
the previously characterized defects in the lytic activity of NK
cells, these defects could explain the clinical manifestations of XLP
and related disorders resulting from SAP deficiency.
4389
Acknowledgments
We thank the patients, their families, and the medical staff that took care
of them. We are grateful to Dr Nichols for the anti-SAP Abs, and to Dr
Aversa for the anti-SLAM mAbs.
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2005 105: 4383-4389
doi:10.1182/blood-2004-08-3269 originally published online
January 27, 2005
SAP controls the cytolytic activity of CD8+ T cells against EBV-infected
cells
Loïc Dupré, Grazia Andolfi, Stuart G. Tangye, Rita Clementi, Franco Locatelli, Maurizio Aricò,
Alessandro Aiuti and Maria-Grazia Roncarolo
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