Published online before print December 26, 2008
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,1
Departments of
* Immunology and
Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
2 Correspondence: Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. E-mail: xiongwenwu{at}hotmail.com
ABSTRACT
The graft-versus-leukemia effect of allogeneic marrow transplantation suggests the dramatic effect of the allogeneic T cell to eradicate malignant disease. Preparation and adoptive transfusion of tumor-specific T cells from HLA-mismatched donors might be expected to circumvent CTL tolerance to the tumor. In this study, a soluble, divalent HLA-A2 molecule was constructed with the Fc part of human IgG1 and was pulsed with a peptide related to melanoma tyrosinase 368–376 [Tyr368–376 (Tyr)] to form the Tyr/HLA-A2 dimer, which allowed loading onto monocytes via interaction of the Fc and FcR. The HLA-A2-negative (HLA-A2-ve) monocytes loaded with the Tyr/HLA-A2 dimer acted as allo-APC with copies of a single allogeneic epitope. After coculture of the HLA-A2-ve PBLs and autologous monocytes loaded with the dimer, CD8+ cells in the coculture show an obvious proliferation and increased frequency of Tyr/HLA-A2 tetramer-stained cells. The sorted Tyr/HLA-A2 tetramer-positive CD8+ cells display an elevated cytotoxic activity against HLA-A2-positive melanoma cells expressing tyrosinase endogenously (i.e., SK-Mel-5) but little against tyrosinase-negative melanoma cells (i.e., A375). The coculture of PBLs and autologous monocytes loaded with allogeneic peptide/HLA complexes offers a novel approach to expand allo-restricted, peptide-specific CTLs, which might be a potential arsenal for treatment of patients with malignant disease, if the tumor-related epitope were defined.
Key Words: HLA-A2 dimer • allo-antigen-presenting cells • allo-restriction • T cells
INTRODUCTION
It is well-established that leukemia patients can benefit from a graft-versus-leukemia reaction (GVLR), whereby donor T lymphocytes mount an immune response against recipients’ leukemia cells [1 , 2 ]. Based on it, many efforts are being done to raise allo-restricted CTLs against cellular protein that is overexpressed in tumors, which is called graft-versus-tumor reaction (GVTR) [3 , 4 ]. As a large proportion of tumors expresses elevated levels of normal self-proteins, which are expressed ubiquitously, the specific T cell clones are likely to be deleted from an autologous repertoire [5 , 6 ]. Researchers have been trying to circumvent the T cell tolerance by exploiting the T cell repertoire of MHC-mismatched responders. However, GVLR is associated with graft-versus-host disease (GVHD) in most cases [7 ].
Recent evidences indicate that T cell alloresponse involves recognition of self-peptide antigens resident in the MHC molecule, which comes from work showing that certain murine alloreactive T cell clones can lyse human cells transfected with the appropriate murine class I MHC molecule only when peptides derived from murine cell lines are added [8 , 9 ]. In a previous study, we also showed that self-peptide binding to the HLA-A2 molecule can stimulate HLA-A2-negative (HLA-A2-ve) PBLs and induce a peptide-specific, allogeneic T cell response in vitro [10 ]. As alloreactive CTLs are able to recognize the peptide/MHC combination in a peptide-dependent way, generation and adoptive transfusion of peptide-specific, alloreactive CTLs are expected to mediate GVTR without GVHD in host. However, in a real case of bone marrow transplantation or coculture of tumor cells with allogeneic lymphocytes, a different set of "irrelevant" endogenous peptides derived from various components of allogeneic stimulator cells would occupy peptide-binding grooves of the MHC. It may lead to potential stimulation of CTLs for various allogeneic epitopes, which makes the isolation of peptide-specific CTLs unsuccessful [11 ]. Furthermore, these CTLs with multiple specificities would account for various tissue injuries observed in GVHD.
In this study, a divalent HLA-A2/IgG1-Fc molecule (HLA-A2 dimer) is constructed by fusing the extracellular domains of HLA-A2 with the constant domains of human IgG1, which is designed to consist of divalent TCR ligands and a Fc part of human IgG1. The molecule is able to attach the TCR ligands to the cells bearing Fc
RI, such as monocytes [12
]. Although the similar strategy has been reported to generate nominal, single epitope-specific CTLs [13
, 14
], we explore the feasibility of this strategy to generate allo-restricted, peptide-specific CTLs. Via interaction of the Fc and FcR, the peptide/HLA-A2 complex can be introduced as an allogeneic epitope onto monocytes of the HLA-A2-ve donor. For the HLA-A2-ve lymphocyte sample and its autologous monocytes, the peptide/HLA-A2 complex on monocytes can be recognized by HLA-A2-restricted, alloreactive CTLs [15
]. After the coculture in vitro, the dimer-loaded HLA-A2-ve monocytes promote autologous PBLs proliferating, and the expanded CD8 + T cells show a peptide/HLA-A2-specific cytotoxicity (Fig. 1
).
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Figure 1. Procedures for generation of allo-restricted CTLs by coculture of HLA-A2-ve PBLs and autologous monocytes loaded with the HLA-A2 dimer. Step 1, preparing the peptide/HLA-A2 dimer and loading the dimer onto HLA-A2-ve monocytes; step 2, coculturing of the peptide/HLA-A2 dimer-loaded monocytes with autologous PBLs; step 3, proliferation and specific tetramer staining assay for CD8+ T cells in the coculture bulk; step 4, sorting and cytolytic assay for the peptide/HLA-A2-specific T cell.
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Cell lines, peptide, and target cells
721.221 is a HLA class I antigen (HLA-A, -B, or -C)-deficient B-lymphoblastoid cell line that expresses a high level of β2 microglobulin (β2m) [16
]. The 1 heavy chains in 721.221 are diminished by 98%, and cell-surface-associated IgG and secreted IgG are absent [17
]. T2 is a transporter associated to antigen processing (TAP)-deficient human cell line and accordingly, expresses reduced amounts of HLA-A2 and no other HLA allele on the cell surface [18
, 19
]. A375 and SK-Mel-5 are HLA-A2-positive (HLA-A2+ve) melanoma cell lines, but tyrosinase is reported to be expressed only by SK-Mel-5 [6
, 20
].
HLA-A2-restricted peptides of self-protein origin tyrosinase 368–376 {Tyr368–376 (Tyr; YMDGTMSQV) [21 ]}, EBV origin latent membrane protein 2A (LMP2A)426–434 (CLGGLLTMV) [22 ], and HIV origin P17 Gag protein Gag77–85 (SLYNTVATL) [23 ] were synthesized by a peptide synthesizer and purified to >90% homogeneity by reverse-phase HPLC. Tyr is a HLA-A2-restricted peptide from membrane-associated protein tyrosinase related to melanoma [21 ]. Gag77–85 peptide is used as a nonspecific control in this study. The peptides were dissolved in DMSO and diluted to 1 mg/ml with RPMI-1640 medium. T2 cell pulsed with Tyr or Gag77–85 is named T2/Tyr or T2/HIV, respectively. A375 pulsed with Tyr is named A375/Tyr.
Construction and expression of the divalent HLA-A2/IgG1-Fc molecule
The cDNA coding for the extracellular domains (
1, 2, 3) of HLA-A*0201 and the cDNA for the Fc fragment (CH1-hinge-CH2-CH3) of human IgG1 were cloned by RT-PCR from T2 cells and PBMC of a healthy donor, respectively. The cloned cDNA were recombined into pCDNA3.1+ vector, generating the pCDNA3.1 + (HLA-A2/IgG1) plasmid. For expression of the divalent HLA-A2 molecule, the plasmid was transfected into the 721.221 with LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA; Cat. No.11668-027) according to the manufacturer’s instructions. Transfected 721.221 cells were selected with G418 (800 µg/ml, Sigma Chemical Co., St. Louis, MO, USA; Cat. No. A1720) for 
20 days until the G418-positive clones expanded. The potential positives were cloned by a limited dilution method in 96-well plates for 15 days, followed by detection of the HLA-A2 dimer expression. There were at least six stable expression clones achieved from 51 clones. The most effective expression clone is named as dimer 721.221, which was used in this study. Until now, over 50 passages of the dimer 721.221 have been achieved, and it can be frozen and recovered without losing the expression character.
Detection of the HLA-A2 dimer by ELISA and Western blotting
The HLA-A2 dimer secreted by the pCDNA3.1 + (HLA-A2/IgG1)-transfected 721.221 cell was detected by ELISA with HLA class I molecule-specific and human IgG1-Fc-specific antibodies. Briefly, 96-well, flat-bottom plates were coated with W6/32, which is a conformation-dependent, anti-HLA class I mAb [24
], and supernatants from pCDNA3.1 + (HLA-A2/IgG1)-transfected 721.221 clones were added, followed by addition of the HRP anti-human IgG1-Fc antibody (Pierce, Rockford, IL, USA; Cat. No. 31413). Plates were washed and developed with o-phenylenediamine dihydrochloride substrate for 5–10 min. The reaction was stopped with the addition of H2SO4 (2 M), and absorbance was read at 490 nm (A490).
The HLA-A2 dimer was run on 10% polyacrylaminde gels containing SDS, with or without the treatment of 2-ME. The separated proteins by the SDS-PAGE were electrotransferred to a nitrocellulose membrane and visualized by HRP anti-human IgG1-Fc antibody.
Preparation of the peptide/HLA-A2 dimer
The dimer 721.221 cells were expanded largely with RPMI-1640 medium containing 10% FCS. The cells were harvested by centrifugation and placed in RPMI-1640 medium without serum at 1 x 106/ml. After incubation at 37ºC, 5%CO2 for 24 h, the supernatants were harvested and concentrated by centrion-10 filtration. The concentrated supernatants containing the HLA-A2 dimer were pulsed with peptide at 20 µg/ml by gentle rotation at 4ºC for 24–48 h. The HLA-A2 dimer pulsed with Tyr or LMP2A426–434 is named the Tyr/HLA-A2 dimer or LMP/HLA-A2 dimer, respectively. The empty dimer without associated peptide was prepared by treating the dimer at pH 3.3 with citrate-phosphate buffer to dissociated the peptide from the HLA-A2-binding grove [25
].
The concentration of the peptide/HLA-A2 dimer was estimated by ELISA with W6/32 and rabbit anti-human β2m antiserum (Beckman Coulter, Fullerton, CA, USA; M202302). The soluble HLA-A2 molecule (1 mg/ml; refolded in vitro, as described previously [26 ]) was used for reference.
IgG1-Fc and FcRI-binding assay and loading peptide/HLA-A2 dimer onto monocytes
Samples of peripheral blood were obtained from three healthy HLA-A2-ve donors. HLA typing was performed with sequence-specific primers [27
]. The Ethical Committee of Tongji Medical College (China) approved sampling of the blood, and informed consent was obtained from all subjects. PBMC was isolated by density gradient centrifugation (Ficoll-Hypaque density 1.077 g/ml), washed twice in sterile PBS, and suspended in 10% FCS RPMI-1640 medium and then placed in tissue-culture dishes for separation into adherent and nonadherent populations by incubation for 1 h. The nonadherent cells (i.e., PBLs) were separated for in vitro immunization protocol. The adherent cells (i.e., monocytes) were treated for 15 min with a 1:1 mixture of 10% FCS RPMI-1640 medium and 10 mM EDTA in PBS to release the monocytes as described [28
]. Monocytes obtained were washed with 10% FCS RPMI-1640 medium (adjust pH to 6.0 with 1 M HCl) to remove the spontaneously bound IgG1 and then incubated with reference IgG1 (purified from human serum by protein A affinity chromatography) or the dimer and detected by anti-human IgG1 antibody or HLA-A2 allele-specific mAb BB7.2 accordingly.
In vitro immunization protocol
The dimer was loaded onto HLA-A2-ve monocytes by incubating the monocytes with 1 µM dimer in PBS at 37ºC, 5% CO2 for 1 h. The dimer-loaded monocytes were plated into 24-well plates (2x105 cells/well). Autologous PBLs (HLA-A2-ve) were stained with CFSE and plated into 24-well plates (1x106 cells/well). Coculture was established at the ratio of five PBLs to one peptide/HLA-A2 dimer-loaded monocyte in 10% FCS RPMI-1640 medium.
T lymphocyte proliferation assay
On Day 6, cells of the coculture were harvested by centrifugation and incubated on ice with PEcy5-conjugated anti-CD8 antibody (anti-CD8-PEcy5; Becton Dickinson, Heidelberg, Germany; Cat. No. 555636). After 1 h incubation, cells were washed extensively and then fixed with PBS containing 2% formaldehyde. Double-color analysis was performed with CFSE and anti-CD8-PEcy5 using a FACSCalibur (Becton Dickinson). Flow cytometric data files were analyzed with the "Proliferation Wizard" module in ModFit LT Macintosh software [29
, 30
].
Specific tetramer-staining assay
PE-labeled Tyr/HLA-A2 tetramer or LMP/HLA-A2 tetramer was prepared by mixing the biotinylated Tyr/HLA-A2 or LMP/HLA-A2 complex with streptavidin-PE as described previously [26
, 31
]. On Day 10, cocultural bulk was incubated on ice with 10 µg/ml tetrameric complexes. After 30 min incubation, cells were washed extensively with PBS containing 1% FCS. Anti-CD8-PEcy5 antibody was added subsequently, and the samples were incubated on ice for a further 30 min. After extensive washing, samples were fixed with PBS containing 2% formaldehyde. Double-color analysis was performed with tetramer-PE and anti-CD8-PEcy5. HIV/HLA-A2 tetramer was also prepared in the same way as nonspecific-binding control.
Enrichment of Tyr/HLA-A2-specific T cells
For subsequent tetramer sorting, CD8+ T cells were enriched by one round of positive immunomagnetic sorting with BD IMag anti-human CD8 particle (Becton Dickinson, Cat. No. 557812), following the protocol recommended by the manufacture, and then cultured one more week with the Tyr/HLA-A2 dimer-loaded monocytes. Streptavidin-coated beads (Becton Dickinson, Cat. No. 557812) were used for enrichment of Tyr/HLA-A2 tetramer-positive cells. The beads were incubated for 30 min with biotinylated Tyr/HLA-A2 monomers (50 µl at 10 µg/ml for 1x107 beads) and mixed with CD8+ T cells. After they were sorted immunomagnetically, bead-coated cells were then expanded for 1 week with the Tyr/HLA-A2 dimer-loaded monocytes and recombinant IL-2 and tested for cytotoxicity.
Cytotoxicity assay
Cytotoxicity assay of the sorted, Tyr/HLA-A2-specific T cells was performed with the nonradioactive enzyme immunoassay using CytoTox 96 (Promega, Madison, WI, USA; Cat. No. G1780). The irradiated target cells were added to round-bottom 96-well plates at a final concentration of 2 x 104 cells/well. Cocultural bulk as added at an E:T ratio of 2:1 and incubated for 4 h at 37ºC. Specific lysis was calculated as (experimental release–spontaneous release)/(maximum release–spontaneous release) x 100%.
Statistical analysis
Statistical analysis among experimental groups was performed by ANOVA. A value of P < 0.05 was considered statistically significant. The significant difference were marked as *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; and ***, P < 0.001.
RESULTS
Preparation of the divalent HLA-A2/IgG1-Fc molecule (HLA-A2 dimer)
To determine whether the pCDNA3.1 + (HLA-A2/IgG1)-transfected 721.221 cell is able to produce the HLA-A2 dimer of proposed conformation, culture supernatants were detected by HLA class I molecule-specific (W6/32) and IgG1-Fc-specific antibodies in a sandwich ELISA. As shown in Figure 2A
, supernatants of the dimer 721.221 are reactive with the W6/32 and anti-human IgG1-Fc, and supernatants of untransfected 721.221 cells are not. As the W6/32 is conformation-dependent, binding of the antibody to the product suggests that the HLA-A2 dimer is conformationally intact. Western blot analysis of nonreduced SDS-PAGE with anti-human IgG1-Fc shows a protein 150 kD expressed in dimer 721.221, which reduced to 70 kD, suggesting that the protein is in a divalent form (Fig. 2B)
. Dimer 721.221 secrets 20 nM HLA-A2 dimer/106 cells/day in RPMI-1640 medium without FCS. The HLA-A2 dimer was concentrated to 1 µM for the following study.
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Figure 2. (A) Detection of the dimer by sandwich ELISA with W6/32 and HRP anti-human IgG1-Fc antibodies. Supernatants of the dimer 721.221 were diluted by 1:10. The RPMI 1640 and supernatants of untransfected 721.221 cells were tested as negative control. Each sample was assayed in nonuple, and the results represent the mean value of A490 ± SD with a marker showing the difference between groups (***, P<0.001). The result shows the expression product is reactive with HLA class I-specific and anti-human IgG1-Fc antibodies. (B) Western blot analysis of the dimer with anti-human IgG1-Fc antibody. Lane 1, Reduced SDS-PAGE; lane 2, nonreduced SDS-PAGE. Supernatants of the transfected cells show a protein 70 kD in reduced SDS-PAGE and 150 kD in nonreduced SDS-PAGE.
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RI constitutively presents monocytes as the high-affinity receptor for IgG1, which is the only type of FcR able to bind monomeric IgG1 (opposite to immune complexes) [32
]. The binding dynamics were observed by using the reference IgG1. As shown in Figure 3A
, IgG1 can be loaded onto monocytes efficiently in a concentration-dependent way. The binding of the reference IgG1 to monocytes is significant when the concentration is 1 µM and reaches maximum binding at 8 µM. Although monocyte-associated IgG1 decreases rapidly in 1 h, it increases slowly after 4 h and remains associated for days. The adding of monensin, which is an inhibitor of receptor-mediated internalization, does not improve the amount of IgG1 associated on monocytes but inhibits the latter increase (Fig. 3B)
. Binding efficiency of the Tyr/HLA-A2 dimer to monocytes is detected by measuring the amount of the Fc fragment (Fig. 3C)
or HLA-A2 (Fig. 3D)
on the HLA-A2-ve monocytes. The Tyr/HLA-A2 dimer can be loaded efficiently onto the monocytes, and the HLA-A2-ve monocytes display a significant amount of HLA-A2 molecules on the cell surface after loading. A similar result was observed for the LMP/HLA-A2 dimer (data not shown).
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Figure 3. (A) IgG1 can bind to FcRI in a dose-dependent way. The monocytes were incubated with IgG1, varied of different concentration, and detected by FITC anti-Fc antibody. The data are shown as the mean ± SD mean fluorescence intensity (MFI) from three separate experiments. (B) Receptor-bound IgG1 remains cell-associated at 37ºC for days. Cells were detected by FITC anti-IgG1-Fc antibody. Monocytes without IgG1 loaded ( ) show low MFI, whereas monocytes loaded with 8 µM IgG1 (x) remain with a relatively high MFI in a variety of times. The adding of monensin ( ) does not increase the amount of IgG1 associated on monocytes. The data are shown as the mean ± SD MFI from three separate experiments. (C) Comparing the fluorescence of monocytes alone (peak a), monocytes loaded with 1 µM Tyr/HLA-A2 dimer (peak b), and monocytes with 8 µM reference IgG1 (peak c) detected by FITC anti-IgG1-Fc antibody. (D) Monocytes alone (left) or monocytes loaded with 1 µM Tyr/HLA-A2 dimer (right) are detected by HLA-A2-specific mAb BB7.2. HLA-A2-ve monocytes display significant amounts of HLA-A2 on the surface after loaded with the Tyr/HLA-A2 dimer. FSC-A, Forward-scatter A.
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Figure 4. PBL samples of three HLA-A2-ve individuals (Donors 1, 2, and 3) were stimulated by autologous monocytes loaded with the peptide/HLA-A2 dimer. The PI and percentage of proliferated cells of CD8+ cells were tested by CFSE dilution assay. (A) The HLA-A2-ve CD8+ T cells show an elevated proliferation after stimulating by autologous monocytes loaded with the Tyr/HLA-A2 dimer (d) or LMP/HLA-A2 dimer (e), compared with that by monocytes alone (a, the negative control), monocytes plus Tyr (b), or monocytes loaded with empty dimer (c). The proliferated cells are seen as the subpopulation with lower CFSE intensity (M2) compared with the parent cells without proliferation (M1). Various peaks with different gray represent generations of the proliferated cells. The cytometric data show the results of Donor 1. (B) The mean values of PI ± SD of three HLA-A2-ve individuals are represented with markers showing the difference of other groups compared with the negative control. (C) The mean values of percentages of proliferated cells ± SD of three HLA-A2-ve individuals are represented with markers showing the difference of other groups compared with the negative control (*, 0.01<P<0.05; ***, P<0.001).
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Figure 5. The PBL samples of three HLA-A2-ve individuals (Donors 1, 2, and 3) were stimulated by autologous monocytes loaded with the Tyr/HLA-A2 dimer (upper panel) or LMP/HLA-A2 dimer (lower panel). The PBLs stimulated by autologous monocytes without the dimer loaded were as negative control. Tyr/HLA-A2 tetramer (upper panel) or LMP/HLA-A2 tetramer (lower panel) was used to stain the corresponding bulk, and an irrelevant HIV/HLA-A2 tetramer was used as nonspecific binding control. On Day 10 of the coculture, the bulks were stained with the anti-CD8-PEcy5 and tetramer. (A) Percentages of tetramer-positive CD8+ cells of samples of three individuals stimulated by Tyr/HLA-A2 dimer-loaded monocytes are presented. (B) The mean values of percentage of tetramer-positive CD8+ cells ± SD of samples of three individuals stimulated by Tyr/HLA-A2 dimer-loaded monocytes are represented with a marker showing the difference between groups. (C) Percentages of tetramer-positive CD8+ cells of samples of three individuals stimulated by LMP/HLA-A2 dimer-loaded monocytes are presented. (D) The mean values of percentage of tetramer-positive CD8+ cells ± SD of samples of three individuals stimulated by LMP/HLA-A2 dimer-loaded monocytes are represented with a marker showing the difference between groups (*, 0.01<P<0.05; **, 0.001<P<0.01).
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50% Tyr/HLA-A2 tetramer-stained CD8+ cells (data not shown). However, the sorted cells from samples of three HLA-A2-ve individuals display a marked, specific cytolytic activity against target T2 cells pulsed with appropriate Tyr peptide (T2/Tyr) in an E:T ratio of 2:1 but a backgroud killing against T2 cells loaded with irrelevant peptide origin from HIV (T2/HIV; Fig. 6
). Interestingly, cytotoxicity of the sorted cells against two HLA-A2+ve human melanoma cells, SK-Mel-5 and A375, is different. The tyrosinase-expressing SK-Mel-5 melanoma cells can be killed efficiently, but tyrosinase-negative A375 melanoma cells cannot be, and the cytotoxicity can be restored after A375 pulsing with the Tyr peptide (A375/Tyr; Fig. 6
). The unsorted, cocultural bulk exhibits a similar pattern of cytolytic activity to these targets at a higher E:T ratio of 20:1 (data not shown). These findings indicate that the tetramer-positive CD8+ cell expended in the coculture bears a peptide-specific CTL activity against targets carrying appropriate ligand, including human tumor cells.
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Figure 6. Tyr/HLA-A2 tetramer-positive CD8+ cells expanded by Tyr/HLA-A2 dimer-loaded monocytes were enriched through immunomagnetic sorting using biotinylated Tyr/HLA-A2 monomers bound to streptavidin-coated beads. The enriched cells were analyzed for their cytotoxicity against a panel of target cells. The cytolytic activity against the irrelevant target cells (T2/HIV) was used as negative control. (A) The specific lysis for the samples of three HLA-A2-ve individuals (Donors 1, 2, and 3). (B) The mean values of specific lysis ± SD of three individuals are represented with markers showing the difference between the negative control and other groups (***, P<0.001). The enriched Tyr/HLA-A2 tetramer-positive CD8+ cells exhibit an elevated cytotoxicity against tyrosinase-expressing SK-Mel-5 melanoma cells compared with that against tyrosinase-negative A375 melanoma cells (29.07%±2.68% vs. 11.73±4.86, P=0.011).
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In contrast to nominal antigen-induced immunity, the immune response to tumor is often less vigorous and results in CTLs of lower avidity, especially when tumor antigens are represented by normal self-proteins [33 , 34 ]. The general strategy of using peptide-pulsed APCs to generate tumor antigen-specific CTLs preferentially enriches a population of low-affinity, peptide-specific CTL clones that do not recognize tumor endogenously expressing antigens [5 , 35 ]. Furthermore, studies have demonstrated that the loss of high-avidity CTLs following in vitro stimulation with peptide-pulsed APCs makes the isolation of tumor-reactive CTLs from patients more difficult [36 , 37 ]. As many evidences show, most alloreactive T cells clones are likely peptide-specific [8 9 10 ]. Many efforts have being done to raise allo-restricted CTLs against cellular protein that is overexpressed in tumors. However, allogeneic stimulator cells would present a different set of irrelevant, endogenous peptides, leading to potential stimulation of CTLs for the various allogeneic epitopes. Although the TAP-deficient T2 cell line might be used for this purpose, irrelevant peptides occupying the groove of the HLA-A2 molecule cannot be excluded completely for TAP-independent pathway [19 ]. To make APCs "present" a single allogeneic epitope, a divalent HLA-A2/IgG1-Fc molecule constructed in this report contains a Fc fragment of human IgG1 and divalent TCR ligands, which is able to bind to monocytes and cross-link specific TCRs of T cells. For HLA-A2-ve lymphocytes, the peptide/HLA-A2 complex of the dimer loaded onto autologous monocytes would be a sole alien antigen, which is expected to induce autologous PBLs to generate HLA-A2-restricted, peptide-specific CTLs in the coculture in vitro.
Although a soluble, divalent MHC molecule is reported to induce nominal, antigen-specific CTLs, the induction efficiency is not satisfactory. There are studies showing that a soluble, divalent H-2Kb/IgG1 molecule, which was generated by fusing the extracellular domains of murine H-2Kb to the constant domains of the murine IgG1, could stimulate an antigen-specific T cell and up-regulate CD69 expression on the surface of CD8+ T cells. However, full T cell activation was not achieved [13
, 14
]. The reason for the observation would be the low affinity of mouse IgG1 binding to FcRI, which is the only type of FcR that binds monomeric IgG (preferentially, murine IgG2a and human IgG1) [38
]. The Fc part of murine IgG1 binds to FcRI with affinity 100- to 1000-fold lower than that of the murine IgG2a. The inefficient binding of a soluble H-2Kb/IgG1 molecule to FcRI-expressing cells may result in the induction of T cell anergy instead of activation, for lack of costimulatory signals provided by FcRI-expressing cells [39
, 40
].
The aim of this study is to show experimental evidence that an allogeneic MHC molecule associated with its restricted peptide, attached to monocytes cocultured with autologous PBLs, is able to generate the peptide/allo-MHC-specific T cells. The divalent HLA-A2/IgG1-Fc molecule used in this study contains the CH1 and hinge region in the Ig scaffold of the dimer, which provides more stable and flexible access for TCR binding. The Fc domain of human IgG1 makes the dimer able to bind FcRI-bearing cells, such as monocytes from peripheral blood [12
, 38
]. As the only type of high affinity for monomeric IgG (opposite of immune complexes), FcRI constitutively present on monocytes with Kd = 10–8 M for monomeric, human IgG1. Scatchard plots of IgG1 binding shows 18,000 binding sites/cell on the monocyte surface [32
]. Unlike the IgG-containing immune complexes, the binding of FcRI with monomeric IgG1 stimulates little endocytosis [28
], or the noncross-linked FcRI-IgG1 is internalized and recycled rapidly to the cell surface without entering the endocytic pathway [41
]. Our results show that monocyte-associated IgG1 decreases rapidly and increases slowly, and adding of monensin inhibits the latter increase, which suggests the existence of internalizing and recycling FcRI with the ligand still bound. These characteristics enable IgG1 to bind to monocytes efficiently and remain membrane-associated on the surface of monocytes for a relatively longer time. Binding of ligand to FcR also promotes multivalent cross-linking of TCR and delivering of costimulatory signals [14
], which are necessary for full T cell activation.
In this study, PBL samples of three HLA-A2-ve individuals were stimulated by autologous monocytes loaded with the Tyr/HLA-A2 dimer. After coculture, the bulk CD8+ cells show increased proliferation as well as elevated frequency of staining with a specific Tyr/HLA-A2 tetramer. A cytotoxicity assay reveals that the generated CTLs are Tyr/HLA-A2-specific. The specificity of the generated CTLs was further tested by using two HLA-A2+ve melanoma cell lines as targets: tyrosinase-expressing SK-Mel-5 and tyrosinase-negative A375. The sorted Tyr/HLA-A2 tetramer-positive CD8+ cells from the coculture kill the SK-Mel-5 cells much more efficiently than the A375 cells; the cytotoxicity against the A375 can be restored after peptide Tyr is pulsed, demonstrating that allogeneic, divalent peptide/HLA class I complexes attached to monocytes can stimulate autologous PBLs to generate allo-restricted, peptide-specific CTLs in vitro. This study is expected to provide a strategy in vitro to generate peptide-specific CTLs from HLA-mismatched donors for tumor-adoptive immunotherapy to circumvent host T cell tolerance to tumor. Apparently, as the CTLs generated are specific for allogeneic HLA associated with its restricted peptide, and no cells in the host express an allogeneic HLA molecule spontaneously, it would be inappropriate to use an allogeneic peptide/HLA dimer for in vivo immunization, which is different from the strategy using syngeneic peptide/MHC dimers as a vaccine to immunize mice [13 , 14 ]. Compared with autologous CTL transfusion, the allogeneic CTLs might contain alloreactive T cell clones with multiple specificities, which would incur various tissue injuries. Therefore, before adoptive transfusion, the allogeneic CTLs generated must be well-sorted. Another issue is that the host would reject allogeneic CTLs, which would abolish the efficacy of adoptive transfusion. The gap between the CTL killing target (within minutes) and the host rejecting the CTLs (in a couple of days) would provide a chance for allogeneic CTLs to carry out their function as well as an opportunity to terminate the CTLs efficacy. However, future studies exploring the human/SCID mouse melanoma model are required to show in vivo capacity of the generated CTLs to kill tumor cells and to avoid GVHD injuries. If produced to good manufacturing practices grade, peptide-specific, allogeneic CTLs expanded by the dimers might benefit adoptive treatment for patients with tumors, provided the corresponding epitope is defined.
ACKNOWLEDGEMENTS
This work was supported by grants from the National Natural Science Foundation of China (No. 30772040) and the "973" project of the Chinese Department of Science and Technology (No. 2007CB512900). The authors specially thank Drs. Xueling Chen, Jianan Li, Wei Sun, Yinhong Song, Hongxia Duan, and Qing Li for their kind assistance with this work.
FOOTNOTES
1 These authors contributed equally to this work. 
Received April 15, 2008; revised November 2, 2008; accepted November 10, 2008.
REFERENCES
receptors FASEB J. 4,3275-3283[Abstract] RI-mediated endocytosis J. Biol. Chem. 269,24396-24402
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