ABBS 2005,38(03): Induction of the Epstein-Barr Virus Latent Membrane Protein 2 Antigen-specific Cytotoxic T Lymphocytes Using Human Leukocyte Antigen Tetramer-based Artificial Antigen-presenting Cells

*Corresponding
author: Tel, 86-27-83692611; E-mail, [email protected]

Abstract        Cytotoxic T lymphocytes
(CTLs) specific for the Epstein-Barr virus (EBV) latent membrane protein 2
(LMP2) antigen are important reagents for the treatment of some EBV-associated
malignancies, such as EBV-positive Hodgkin’s disease and nasopharyngeal
carcinoma. However, the therapeutic amount of CTLs is often hampered by the
limited supply of antigen-presenting cells. To address this issue, an
artificial antigen-presenting cell (aAPC) was made by coating a human leukocyte
antigen (HLA)-pLMP2 tetrameric complex, anti-CD28 antibody and CD54 molecule to
a cell-sized latex bead, which provided the dual signals required for T cell
activation. By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral blood
mononuclear cells from HLA-A2 positive healthy donors, LMP2 antigen-specific
CTLs were induced and expanded in vitro. The specificity of the
aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2 tetramer staining and
cytotoxicity against HLA-A2-LMP2 bearing T2 cell, the cytotoxicity was
inhibited by the anti-HLA class I antibody (W6/32). These results showed that
LMP2 antigen-specific CTLs could be induced and expanded in vitro by the
HLA-A2-LMP2-bearing aAPC. Thus, aAPCs coated with an HLA-pLMP2 complex,
anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specific
CTLs for adoptive immunotherapy.

Key words        immunotherapy; cytotoxic T lymphocyte;
artificial antigen-presenting cell; Epstein-Barr virus

The infusion of
cytotoxic T lymphocytes (CTLs) for Epstein-Barr virus (EBV) antigens has been
proved as safe and effective prophylaxis and treatment for EBV-associated
diseases [1–4]. In the
immunocompetent host, EBV is associated with malignancies that express a more
limited array of viral genes. For example, in EBV-positive Hodgkin’s lymphoma
and nasopharyngeal carcinoma, only subdominant EBV antigens, such as latent
membrane protein (LMP) 1 or 2 and Epstein-Barr nuclear antigen 1, are
expressed. Of the potential CTL target antigens expressed in Hodgkin’s lymphoma
and nasopharyngeal carcinoma, LMP2 is the ideal target antigen for
immunotherapy [5,6]. LMP2-specific CTLs can be generated in vitro using
peptide-pulsed autologous dendritic cells (DCs) as antigen-presenting cells
(APCs) [7,8]. However, this method is time-consuming and expensive due to the
limited availability of donor-derived DCs. To date, several reports indicated
effective stimulation of T cells by human leukocyte antigen (HLA)-peptide
ligands coating on artificial antigen-presenting cells (aAPCs), such as
liposomes [9,10] and microbeads [11–14]. The
aAPCs simulate the natural APCs (such as DCs, macrophages and B cells), which
are able to provide the dual signals (the antigen-specific and costimulatory
signals) for T cell activation. These aAPCs can be prepared by coating the
T-cell receptor (TCR) ligand, e.g. peptide-major histocompatibility complex
(MHC), and costimulatory molecules, such as B7, on a cell-sized bead, which
provides a practical and convenient approach to generating antigen-specific T
cells for adoptive immunotherapy.

In this study, we
explored a simple and efficient method to induce LMP2-specific CTLs from the
peripheral blood mononuclear cells (PBMCs) of HLA-A2 positive healthy donors,
using aAPCs prepared by coating an HLA-A2-pLMP2 complex, anti-CD28 antibody and
CD54 molecule to a cell-sized latex microbead in vitro. The interaction
of the T-cell receptor and the HLA-A2-pLMP2 complex provides the basis for
antigen specificity. Signaling through the CD28 receptor provides a powerful
costimulatory signal following engagement of the anti-CD28 antibody. The
adhesion molecule CD54 provides a synergistic signal through the LFA-1 molecule
expressed on T cells. Co-cultured with the aAPCs, the HLA-A2 positive PBMCs can
be induced to generate LMP2-specific CTLs, which bear the antigen-specific
cytolytic properties.

Materials and Methods

Cell line

T2 cell line was kindly
provided by Prof. Nicholas ZAVAZAVA ( 

Synthetic peptides

The following
HLA-A2-restricted peptides were used in this study: the LMP2-derived peptide CLGGLLTMV (referred to as pLMP2) [15], the tyrosinase
peptide YMDGTMSQV (pTyr) [16], and the HIV-Gag peptide SLYNTVATL (pHIV) [17].
Peptides were synthesized by standard solid-phase chemistry and characterized
by mass spectrometry. The purity of the synthetic peptides was more than 90% as
indicated by analytical HPLC. Lyophilized peptides were dissolved in
dimethylsulfoxide and stored at –80 ºC after dilution in phosphate-buffered
saline (PBS).

Preparation of
biotinylated HLA-A2-pLMP2 monomeric and tetrameric complexes

Synthesis of monomeric
and tetrameric HLA-A2-pLMP2 complexes was carried out according to the protocol
of Altman et al. [18]. Briefly, plasmids encoding HLA-A*0201 heavy chain
molecule with a C-terminal biotinylation site and human b 

Generation of aAPCs
bearing HLA-A2-pLMP2

Five microliters of
sulfate polystyrene latex beads (Interfacial Dynamics, Portland, USA) were
incubated sequentially with streptavidin (1 mg/107 beads; Sigma), CD28-specific antibody and
CD54 molecule (1 mg/107 beads and 1.5 mg/107 beads, respectively; BD PharMingen, San
Diego, USA) for 30 min in 1 ml PBS at 4 ºC on a rotator. The beads were then
incubated with biotinylated HLA-A2-pLMP2 monomer (2 mg/107 beads) for 1 h at 4 ºC on a rotator, which
allowed the biotinylated HLA-A2-pLMP2 monomer to bind to the streptavidin
coated on the surface of the beads. After each incubation step, these aAPC
beads were washed with PBS, and stored in the PBS at 4 ºC.

In vitro CTL
induction by co-culture of HLA-A2+PBMCs and aAPCs

PBMCs from healthy
HLA-A2 positive donors were separated using standard Ficoll-Hypaque (Sigma)
gradient density centrifugation. These PBMCs were used as responder cells (3´106 cells/well) and co-cultured with the
HLA-A2-LMP2-bearing aAPCs (3´105 cells/well) in 24-well
plates in RPMI 1640 medium supplemented with 10% FBS (1 ml/well). IL-7 (10
ng/ml) was added on day 1. IL-2 (50 u/ml; R&D Systems,  

Tetramer staining

Tetramer staining was
performed as previously described [19,20]. In brief, 1´106 cells were incubated in 100 ml fluorescence activated cell sorter (FACS)
staining buffer (PBS supplemented with 1% BSA and 0.05% NaN3) with 20 mg/ml HLA-A2-peptide tetramer at 37 ºC for 30 min.
Cells were washed with PBS and subsequently incubated with PE-Cy5 labeled
anti-CD8 antibody (BD PharMingen, San Diego, USA) at 4 ºC for 30 min. All cells
were washed with PBS twice after being stained, then fixed in 1% formaldhyde.
Stained cells were analyzed with FACScalibur (Becton Dickinson).

Cytotoxicity assay

The colorimetric CytoTox
96 assay (Promega,  

Inhibition of the
cytotoxicity with HLA class I-specific monoclonal antibody

T2pLMP2 target cells
were incubated with anti-HLA class I monoclonal antibody W6/32 (ATCC, USA; http://www.atcc.org/) [21] and a control
isotypic monoclonal antibody (mAb) of irrelevant specificity (immunoglobulin G;
BD PharMingen, USA) at a final concentration of 30 mg/ml for 40 min at 4 ºC before cytotoxicity assay. After
incubation, the target cells were mixed with effector cells for the LDH release
assay.

Statistical analysis

All data in this study
were analyzed using SPSS version
10.0 software (SPSS, Chicago, USA). P<0.05 was considered as statistically significant.

Results

Growth kinetics of the
co-culture bulk and the phenotype of the induced T cells

Following the first
stimulation using HLA-A2-pLMP2-bearing aAPCs, PBMCs were expanded continuously.
After 4 weeks of co-culture with aAPCs, an approximately 40-fold increase of
cell number in the culture bulk was achieved. The phenotype of the expanded
cells was measured by flow cytometry. The percentage of CD4+ cells gradually decreased while the CD8+ cells increased with the progression of the
co-culture with aAPCs. The phenotypes of the CTLs for LMP2 were CD8+, CD4–, CD3+, CD16– and CD56–.

Frequency of
LMP2-specific CTLs increased by co-culture with aAPCs as determined by tetramer
staining

Flowcytometric analysis
of PBMCs was performed before or after co-culture with the aAPCs bearing
HLA-A2-pLMP2. Before the four rounds of stimulation using aAPCs, the frequency
of CD8+ T cells stained with HLA-A2-LMP2 tetramers
was 0.07%. However, after stimulation, FACS analysis revealed that 13.9% of CD8+ T cells were stained with HLA-A2-LMP2
tetramers, which was not observed when staining with the control tetramers
(HLA-A2-pHIV tetramer and HLA-A2-pTyr tetramer) (Fig. 1). LMP2-specific
CTLs expanded by aAPCs from the five donors showed similar results (Table 1).

Cytotoxicity of the
aAPC-induced pLMP2-specific CTLs

The cytotoxic activity
of the aAPC-induced CTLs against various target cells (T2pLMP2, T2pHIV, T2pTyr and T2 cells without a
pulsed peptide) was tested using the LDH-releasing assay. The CTLs exhibited
approximately 60% specific lysis against the T2pLMP2 at an effector:target
ratio of 50:1. However, the CTLs showed an approximately 10% cytolysis against
the T2pHIV, T2pTyr and the T2
without a pulsed peptide at the same effector:target ratio (Fig. 2). The
specific killing activity of the CTLs induced by the aAPCs against T2pLMP2
target cells was much more obvious than in any other group (P<0.05). Specific CTLs for LMP2 induced by the aAPCs from the five donors showed similar specific lysis (Table 1). This result shows the cytotoxicity of the
aAPC-induced CTL is pLMP2-specific.

Inhibition of the
cytotoxicity of the aAPC-induced T cells by HLA class I specific mAb (W6/32)

To determine whether the
induced CTLs could recognize the specific target cells in an HLA class
I-restricted manner, anti-HLA class I mAb W6/32 was utilized to block the
cytotoxicity of the aAPC-induced CTLs. The cytotoxic activity against the
T2pLMP2 was significantly eliminated by W6/32. As shown in Fig. 3,
incubation of T2pLMP2 target cells
with W6/32 led to the inhibition of the targeted cells lysis, whereas mouse
immunoglobulin G, used as an isotype control, showed no effect. These results
suggested that the aAPC-induced CTLs lysed the specific targets in an HLA class
I-restricted manner.

Discussion

Adoptive immunotherapy
holds promise as a treatment for cancer. However, nonspecific T cell therapy is
not considered efficient for clinical applications at present because of its low killing activity, lack
of specificity and side-effects. Tumor-specific CD8-positive CTLs constitute
the most important effector cells for antitumor responses [22]. CTLs recognize
“processed” peptides that are derived from endogenous proteins and presented
on the cell surface in association with MHC class I molecules [22,23]. Peptides
that bind to a given MHC class I molecule have been shown to share common amino
acid motifs, which are called major anchor motifs [23]. Hence, tumor-specific
CTLs can recognize and select the antigenic peptides by scanning peptide
sequences, then kill tumor cells in an antigenic peptide-specific fashion. EBV-encoded LMP2 is the target antigen available for therapeutic
augmentation of CTL responses in patients with EBV-associated malignancies
[5,6].

It has been shown
previously that in vitro specific CTLs can be generated using
peptide-pulsed autologous DCs as APCs [24,25]. However, CTL expansion to
clinically relevant amounts requires multiple leukophoreses to obtain enough
autologous DCs. Variability in both quantity and quality of obtained DCs, which
presumably relates to underlying diseases and the pre-treatment of the
patients, also significantly impacts on the viability of DC-based therapeutics.
For these reasons, use of DCs has been a limiting step in ex vivo
expansion of T cells [14]. Strategy of aAPC offers a promising way to overcome
the disadvantages of natural APCs. CTLs generated by artificial stimulation
protocols can kill both peptide-loaded and natural target cells [14,26,27]. The
presented data showed that CTLs specific for LMP2 could be induced in vitro with
aAPCs coating HLA-A2-pLMP2, anti-CD28 antibody and CD54 molecule to cell-sized
latex beads. The HLA-A2 positive PBMCs were induced to generate pLMP2-specific
CTLs in vitro by co-culture with aAPCs, and specificity of the
aAPC-induced CTLs was confirmed by both their binding to HLA-A2-pLMP2 tetramers
and killing activity against HLA-A2-pLMP2-bearing cells. In order to test for
the specificity of the CTL cytotoxicity, T2 cells were used as the target in
this study. The T2 cell is an HLA-A*0201 human lymphoid cell line, which is
defective in endogenous antigen processing and presenting, but can effectively
present exogenously supplied peptides [28,29]. The specific killing activity of
CTLs induced by the aAPCs against specific target cells T2pLMP2 was much more
effective than that of any other control group. The results indicated the
cytotoxicity of the aAPC-induced CTLs is antigen-specific, that is, against the
target cells bearing the corresponding HLA-A2-pLMP2 complex. The aAPC-mediated
stimulation was better than T2pLMP2 stimulation in in vitro expansion of
antigen-specific CTLs, because of the advantages of aAPCs (refer to the
following description).

For clinical studies,
HLA tetramer-based aAPCs have several distinct advantages over cellular APCs,
including DCs. One of these is ease of preparation, which is not required for
sterile cell culture and cytokines, thereby reducing both the variability and expense
associated with ex vivo expansion. The variability is particularly
important when considering therapies for cancer as there have been reported
defects in DCs obtained from patients with malignancies [30]. Another of the
advantages is the good stability of aAPCs, unlike the biologic variability and
patient-to-patient variation when producing cellular APCs such as DCs. In
addition, aAPCs can present defined combinations of MHC alleles and peptides,
and be easily adapted using other MHC alleles and/or peptides, so that
immunodominant or subdominant epitopes can be expanded preferentially. When
cellular APCs are used, an array of MHC molecules is employed, and a broad but
uncontrolled MHC restricted response is generated. The last advantage is the
ability to control the combination of costimulatory complexes associated with
aAPCs, unlike cellular APCs that may participate in the T cell-APC interaction
in such a way to promote tolerance or anergy. For example, on T cell
activation, B7 binding to CTLA-4 instead of CD28 would limit T cell expansion
[31,32]. It is convenient to prepare aAPCs coated with anti-CD28, which binds
specifically to CD28, avoiding the binding of B7 to CTLA-4. In this study, we
used a combination of anti-CD28 and CD54 for the generation of a costimulatory
signal, because CD54 molecule appears to augment cell expansion by limiting
apoptosis when coupled to aAPCs [33].

The use of aAPCs
represents the state-of-the-art in generation of antigen-specific
CTLs for adoptive immunotherapy. Thus, HLA tetramer-based aAPCs coated with an
HLA-peptide complex, anti-CD28 antibody and CD54 molecule could provide a
useful tool for the reproducible expansion of specific CTLs in vitro and
significantly advance the field of adoptive immunotherapy. aAPC might become
useful reagents to enrich LMP2-specific CTLs for the treatment of patients with
EBV-associated malignancies.

Acknowledgements

We thank Dr. Xiao-Bin
JIANG, Dr. Wen-Hua WU and Dr. Guo-An CHEN in the  

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