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Blood, 1 July 2004, Vol. 104, No. 1, pp. 224-226. Prepublished online as a Blood First Edition Paper on March 18, 2004; DOI 10.1182/blood-2003-07-2461.
IMMUNOBIOLOGY Artificial antigen-presenting constructs efficiently stimulate minor histocompatibility antigen–specific cytotoxic T lymphocytesFrom the Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands; Center of Immunology, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis; and Department of Hematology, Leiden University Medical Center, Leiden, the Netherlands.
Cytotoxic T lymphocytes (CTLs) specific for hematopoietic-restricted minor histocompatibility antigens (mHags) are important reagents for adoptive immunotherapy of relapsed leukemia after allogeneic stem cell transplantation. However, expansion of these CTLs to therapeutic numbers is often hampered by the limited supply of antigen-presenting cells (APCs). Therefore, we evaluated whether cell-sized latex beads coated with HLA/mHag complexes HLA-A2/HA-1 or HLA-A2/HA-2 and recombinant CD80 and CD54 molecules can replace professional APCs. The artificial antigen-presenting constructs (aAPCs) effectively stimulated HA-1– and HA-2–specific CTL clones as shown by ligand-specific expansion, cytokine production, and maintenance of cytotoxic activity, without alteration of CTL phenotype. Furthermore, HA-1–specific polyclonal CTL lines were enriched as efficiently by aAPCs as by autologous HA-1 peptide-pulsed dendritic cells. Thus, aAPCs coated with HLA/mHag complexes, CD80, and CD54 may serve as tools for in vitro enrichment of immunotherapeutic mHag-specific CTL lines.
The successful application of donor lymphocyte infusions for the treatment of relapsed leukemia after allogeneic stem cell transplantation illustrates the feasibility of adoptive immunotherapy of hematologic malignancies.1 To minimize graft-versus-host disease, we earlier proposed the use of minor histocompatibility antigens (mHags) HA-1 and HA-2 as immunotherapeutic reagents.2 HA-1 and HA-2 display hematopoietic-restricted tissue distribution and relevant expression on leukemic cells and their progenitors.3-6 Moreover, cytotoxic T lymphocytes (CTLs) directed against these mHags do not cause graft-versus-host disease in an ex vivo skin explant model,7 and coincide with complete remission of relapsed leukemia and multiple myeloma after HLA-matched HA-1– or HA-2–mismatched donor lymphocyte infusions.8 HA-1– and HA-2–specific CTLs can be generated in vitro using peptide-pulsed or mHag-transduced autologous dendritic cells (DCs) as antigen-presenting cells (APCs).9,10 However, expansion of CTLs is difficult due to the limited availability of donor-derived DCs. To date, several reports indicate effective stimulation of T cells by HLA/peptide ligands expressed on artificial antigen-presenting constructs (aAPCs) such as liposomes11,12 or microbeads.13-16 We developed aAPCs that can be manufactured under good manufacturing practice conditions and used to expand mHag-specific CTLs for adoptive immunotherapy. Hereto, cell-sized latex microbeads coated with HLA-A2/HA-1 or HLA-A2/HA-2 complexes, CD80, and CD54 were investigated for their potential to efficiently stimulate HA-1– and HA-2–specific CTL clones and lines while keeping their antigen-specific cytolytic properties.
mHag-specific CTL clones and polyclonal CTL lines, CD4+ T helper cells, and DCs In vivo and in vitro generation of mHag-specific CTL clones, polyclonal CTL lines, and DCs is documented in detail elsewhere.9,17 CD4+ T-helper cells (CD4+Th cells) were generated by culturing peripheral blood mononuclear cells (PBMCs) for 14 days in Iscove modified Dulbecco medium (IMDM) containing 10% pooled human serum (HS) and 0.5% standard Dutch diphtheria/pertussis/tetanus/polio vaccine (National Institute of Public Health and the Environment, Bilthoven, The Netherlands), and 20 U/mL interleukin 2 (IL-2; Cetus, Emeryville, CA) from day 6 onward. Functional assays
All functional assays were performed in IMDM plus 10% HS. Stimulator cells were irradiated (30 Gy) before use. Results of proliferation, interferon-
Proliferation and IFN- Cytotoxicity was evaluated by incubating 2500 51Cr-labeled target cells with serial dilutions of effector CTLs for 4 hours; supernatants were harvested for gamma counting: % specific lysis = (experimental release–spontaneous release)/(maximal release–spontaneous release) x 100%.
Cytokine profiles were tested using the Human Th1/Th2 cytokine cytometric bead assay (BD Biosciences, San Diego, CA). Detection limits were IFN- Synthetic peptides and HLA-A2/mHag complexes HA-1 and HA-2 peptides were synthesized according to the reported sequences.18,19 The biotinylated recombinant HLA-A2/HA-1 and HLA-A2/HA-2 complexes were generated as described and used as monomers for aAPC coating or as tetramers for analysis of HA-1– or HA-2–specific CTLs.20 Ligand immobilization on latex beads The 5.3-µm polystyrene sulfate latex beads (Interfacial Dynamics, Portland, OR) were incubated sequentially with streptavidin-allophycocyanin (1 µg/107 beads; Molecular Probes, Leiden, The Netherlands), with recombinant human CD80/Fc-chimera and CD54 (0.5 µg and 1.5 µg/107 beads, respectively; R&D Systems, Minneapolis, MN), and with 1% human albumin (Sanquin) for 20 minutes in 0.5 mL phosphate-buffered saline (PBS) per 107 beads at 4° C. The beads were then incubated with biotinylated HLA-A2/mHag complexes (0.5 µg/107 beads) for 40 minutes in 1 mL PBS/107 beads at 4° C. After each incubation step, the beads were washed with 1 mL PBS. See the Supplementary Data on the Blood website (click on the "Data Set" link at the top of the online article) for determination of optimal ligand densities.
aAPC-mediated stimulation of mHag-specific CTL clones
The capacity of aAPCs to stimulate mHag-specific CTLs in an efficient and ligand-specific manner was analyzed and compared with that of professional APCs. Three HA-1–specific CTL clones (2.12, 3HA15, 5W38) and 2 HA-2–specific CTL clones (1.7, 1.9) were stimulated with (1) aAPCs coated with the costimulatory molecules CD80 and CD54 only, (2) aAPCs coated with costimulatory molecules and either HLA-A2/HA-1 or HLA-A2/HA-2 complexes, or (3) with HLA-A2+ Epstein-Barr virus (EBV)–transformed B-cell lines naturally expressing HA-1 and HA-2 (HA-1+/HA-2+ EBV-LCLs; Figure 1A-B). All mHag-specific CTL clones showed significant proliferation and IFN-
Improper or partial T-cell receptor signaling by altered peptide ligands or T-cell receptor engagement in the absence of adequate costimulatory signals can lead to changes in cytokine secretion profiles or anergy of T cells.21-23 Similarly, aAPCs may alter the phenotype and function of CTL clones. Therefore, mHag-specific CTL clones (2.12 and 1.7) were stimulated for 7 days with aAPCs or with HA-1+/HA-2+ EBV-LCLs. Subsequently, cytotoxic activity and secretion of IFN- aAPC-mediated enrichment of mHag-specific polyclonal CTL lines The main goal of this study was to investigate whether aAPCs can replace autologous DCs for the enrichment of polyclonal mHag-specific cultures. Hereto, 5 different polyclonal HA-1–specific CTL lines (1, 2, 3, 4, 5), originally induced and restimulated twice with HA-1 peptide-pulsed autologous DCs, were stimulated as follows. CTL lines 1, 2, 3, and 4 were restimulated for 7 days with (1) aAPCs only, (2) aAPCs and autologous PBMCs, (3) aAPCs and autologous CD4+Th cells, or (4) HA-1 peptide-pulsed autologous DCs. HA-1A2 tetramer staining and cytotoxicity assays were performed at day 0 and at day 7 (Table 1). For all CTL lines, similar or improved enrichment for HA-1–specific CTLs was observed after restimulation with aAPCs and autologous feeder cells when compared to DC-mediated enrichment. The feeder cells could either be total PBMCs or activated CD4+Th cells. These responses were driven by antigen presentation via aAPCs because none of the CTL lines showed enrichment after 7 days of incubation with PBMCs or CD4+Th cells only (data not shown). In a separate experiment, CTL lines 4 and 5 were restimulated repeatedly with aAPCs and autologous feeder cells. Both lines showed a 5-fold increase in absolute numbers of HA-1A2 tetramer–positive cells without loss of HA-1–specific cytotoxic activity after 14 days (Figure 2).
In summary, aAPCs coated with HLA-A2/mHag complexes, CD80, and CD54 can be used to selectively enrich mHag-specific CTLs for adoptive immunotherapy. In combination with autologous feeder cells, aAPCs stimulate CTLs as efficiently as peptide-pulsed DCs. The aAPCs can be generated in a nonlaborious way and can be easily removed from the cell culture by a density gradient. All aAPC-constituents are obtainable at clinical grade. Future studies will address the issue of whether aAPCs can be used for the primary in vitro induction of mHag-specific CTLs.
We wish to thank Prof F. Claas and Dr M. J. B. van Stipdonk for critical reading and comments.
Submitted July 21, 2003; accepted February 18, 2004.
Prepublished online as Blood First Edition Paper, March 18, 2004; DOI 10.1182/blood-2003-07-2461.
Supported in part by grants from the Dutch Cancer Society (Koninging Wilhelmina Fonds), the Leukemia and Lymphoma Society, and Foundation `De Drie Lichten.'
The online version of this article contains a data supplement.
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.
Reprints: Liesbeth Oosten, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, E3-Q, Albinusdreef 2, 2333 ZA Leiden, the Netherlands; e-mail: l.e.m.oosten{at}lumc.nl.
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Abstract
Introduction
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