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Autoreactive, Cytotoxic T Lymphocytes Specific for Peptides Derived From Vol. 10, 1047–1056, February 1, 2004 Clinical Cancer Research 1047 Autoreactive, Cytotoxic T Lymphocytes Specific for Peptides Derived from Normal B-Cell Differentiation Antigens in Healthy Individuals and Patients with B-Cell Malignancies Matthias Grube,1 Katayoun Rezvani,1 avidity would be necessary for T-cell immunotherapeutic Adrian Wiestner,2 Hiroshi Fujiwara,1 approaches using the peptides studied. Giuseppe Sconocchia,1 Jan J. Melenhorst,1 1 3 INTRODUCTION Nancy Hensel, Gerald E. Marti, ϩ 2 2 CD8 CTLs specifically recognize peptides derived from Larry W. Kwak, Wyndham Wilson, and endogenous proteins presented by class I HLA molecules on the 1 John A. Barrett surface of malignant cells. T-cell-mediated immune reactions 1National Heart Lung Blood Institute, 2National Cancer Institute, against autologous tumor cells have been described in a variety 3 NIH, and Food and Drug Administration, Bethesda, Maryland of malignant diseases (1–4). Several recent studies have iden- tified tumor-reactive CD4ϩ and CD8ϩ T-lymphocytes in non- Hodgkin lymphoma and chronic lymphocytic leukemia (CLL; ABSTRACT Refs. 5–8). Furthermore, immunoglobulin idiotype vaccination Purpose: To investigate potential immunotherapeutic in myeloma and non-Hodgkin lymphoma can induce immune strategies in B lymphocytic malignancies we looked for responses to malignant B cells (9–12). However normal tissue- CTLs recognizing CD19 and CD20 epitopes. specific self-antigens, which are candidates for immunotherapy Experimental Design: Three CD19 and CD20 peptides in solid tumors (13), have not been widely explored in B-cell binding to HLA-A*0201 were identified and used to detect malignancies (14, 15). T cells reactive to self-antigens are peptide specific CTLs by a quantitative real-time PCR to mainly eliminated by negative thymic selection (16–19). How- ␥ measure IFN- mRNA expression in 23 healthy individuals ever, the process is incomplete, leaving behind low-avidity T and 28 patients (18 chronic lymphocytic leukemia (CLL), 7 cells autoreactive to self-antigens (20, 21). The therapeutic follicular lymphoma, 2 acute lymphocytic leukemia, and 1 potential of monoclonal antibodies targeting the B-cell differ- large cell lymphoma). Peptide-specific CTLs were expanded entiation epitopes CD19 and CD20 makes these molecules of in culture with CD40-activated B cells to test lytic activity in particular interest to study as potential T-cell epitopes (22–26). three patients. T cells, genetically modified to recognize CD19 or CD20 mol- ؉ Results: In healthy individuals, CD8 T-cell responses ecules through antibody ligands can efficiently eliminate their were detected in one to CD1974–82, in three to CD20127–135, respective B-cell target (27–30). Peptides derived from CD20 and three to CD20188–196. Seven of 27 patients (6 with CLL) have been used to generate CTLs to treat B-cell malignancies in ؉ had CD8 T cells recognizing CD1974–82. Seven patients mice (31). CD19 and CD20 play an important role in the responded to CD20127–135 and three to CD20188–196. All development, differentiation, and activation of B cells. CD19 is were CLL patients. CD1974–82-specific CTLs from three a signal-transduction molecule and CD20 is part of a multimeric patients were expanded over 4 weeks. These cells were HLA- receptor complex regulating cell cycle progression and B-cell A*0201 specific and lytic for peptide-loaded antigen- differentiation. Both are expressed by B-progenitor cells, persist presenting cells but not to malignant or unpulsed B cells. during all stages of B-cell maturation, and are lost on terminal Conclusions: CTLs that recognize CD19 and CD20 differentiation to plasma cells (32–34). CD19 and CD20 are epitopes exist in healthy individuals and may be increased largely restricted to normal and neoplastic B cells (although in CLL patients. They are of low avidity and require high CD19 can be also found on dendritic cells). The majority of doses of peptide for activation. Strategies to increase T-cell B-lineage malignancies express CD19 and CD20 (35, 36). These attributes make these molecules good candidates for T-cell immunotherapeutic research. In this study we set out to determine whether cytotoxic CD8ϩ T-cell responses specific for peptides derived from CD19 Received 8/21/03; revised 10/20/03; accepted 10/22/03. and CD20 antigens occur in healthy individuals and patients Grant support: The Dr. Mildred-Scheel-Stiftung Deutsche Krebshilfe Foundation (to M. G.). with B-cell malignancies. We then used a sensitive quantitative The costs of publication of this article were defrayed in part by the real-time (RT) PCR (qPCR) technique to search for the antici- payment of page charges. This article must therefore be hereby marked pated low frequencies of circulating T cells recognizing peptides advertisement in accordance with 18 U.S.C. Section 1734 solely to derived from these molecules. Our results show that low-avidity indicate this fact. Requests for reprints: John Barrett, NIH, 9000 Rockville Pike, Build- T cells recognizing both CD19 and CD20 circulate in normal ing 10, Room 7C 103, Hematology Branch, Bethesda, Maryland 20892. individuals and in patients with B-cell malignancies with a Phone: (301) 402-3296; Fax: (301) 435-8655; E-mail: [email protected]. notable increase in CD19-specific T cells in patients with CLL. Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. 1048 B-Cell Antigen-Specific T Cells MATERIALS AND METHODS zation assay using the T2 cell line as described previously (41, Patients and Donors. After informed consent, cells from 42). T2 cells were pulsed with 100 ␮M peptide and 5 ␮g/ml ␤2 HLA-A2-positive patients with B-cell malignancies [18 CLL, 7 microglobulin (Sigma, St. Louis, MO) for 18 h at 26°C. CMV follicular lymphoma, 2 acute lymphocytic leukemia, 1 large cell (pp65) and gp100 peptides were used as positive controls and lymphoma (non-Hodgkin lymphoma)], which were enrolled in the background expression of HLA-A2 was determined using clinical trials approved by the institutional review board and DMSO as a negative control. The level of stabilized HLA-A2 on healthy individuals (n ϭ 23) were obtained from leucopheresis the surface of T2 was determined by using FITC-conjugated products. Peripheral blood mononuclear cells were separated mouse antihuman monoclonal antibody (BB7.2; BD Bio- using Ficoll-Hypaque gradient-density (Organon Teknika Co., sciences PharMingen, San Diego, CA). The fluorescence index Durham, NC) and were subsequently frozen in RPMI 1640 (FI) determined by fluorescence-activated cell sorting analysis ϭ complete medium (CM; 25 mM HEPES buffer, 2 mML-gluta- for each peptide was calculated by the following formula: FI mine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin; Life (mean channel of fluorescence T2 ϩ peptide)/(mean channel of Technologies, Inc., Gaithersburg, MD) containing 20% heat- fluorescence T2 without peptide). Peptides were considered to inactivated FCS and 10% DMSO according to the standard stabilize HLA-A2 molecules with high affinity, if the FI was protocols. Before use, cells were thawed, washed, and resus- Ն1.5 and low affinity if the FI was between 1.1 and 1.49 at a pended in CM supplemented with 10% human AB serum (HS) peptide concentration of 100 ␮M. All of the assays were re- and rested overnight. peated a minimum of three times, and the results are given as HLA-Typing. High-resolution HLA-class I genotyping means of replicate experiments. ؉ ϩ was performed by sequence-specific PCR using genomic DNA CD8 T-Cell Selection. CD8 T cells were isolated (HLA-Laboratory, Department of Transfusion Medicine, War- from the peripheral blood mononuclear cells of patients and ren G. Magnusson Center, NIH, Bethesda, MD). healthy individuals by using a CD8-positive isolation kit (Dynal, Cell Lines. T2 cells (American Type Culture Collection, Oslo, Norway). After detachment of the immunomagnetic beads ϩ Manassas, VA) are a HLA-A*0201-positive hybrid human cell by using DetachaBead (Dynal) a purity of at last 98% CD8 T line. They expresses very low levels of cell surface HLA-A 2.1 cells was determined by flow cytometry. ؉ and are unable to present endogenous antigens because of the CD8 T Cell in Vitro Stimulation. To determine ϩ lack of most of the MHC class II region including the known peptide-specific CD8 T-cell reactivity, we measured the IFN-␥ ϩ TAP (transporter proteins for antigenic peptide) and proteasome mRNA gene expression by CD8 T cells stimulated with can- genes (37). C1R-A2 cells are a MHC-class I-defective LCL cell didate peptides. Multiple experiments to optimize assay condi- ϩ line, that expresses a transfected genomic clone of HLA-A2.1 tions were performed with CD8 T cells obtained from HLA- (38, 39) and were kindly provided by the laboratory of Dr. A*0201-positive, CMV-seropositive donors, stimulated with Jeffrey Schlom (National Cancer Institute, NIH, Bethesda, MD). HLA-A*0201-associated CMV peptide (pp65)-pulsed C1R-A2 6 ϩ The cells were maintained in CM supplemented with 10% FCS. cells. After purification, 1 ϫ 10 CD8 T cells were plated in a Peptide Predictions. The protein sequence from CD19 96-well flat-bottomed plate in 200 ␮l of CM supplemented with was reviewed for 9-mer peptides that could potentially bind to 10% HS and incubated overnight at 37°C, 5% CO2, to minimize MHC class-I molecules using the computer-based prediction background expression of IFN-␥ mRNA expression due to ϩ analysis of H. G. Rammensee, (University of Tu¨bingen, Tu¨bin- lymphocyte manipulation. CD8 T cells were then stimulated in gen, Germany; Ref. 40).4 In this analysis, peptides are ranked vitro with test peptides using an adapted protocol from previous according to a score that takes the presence of primary and studies (43). Briefly C1R-A2 cells were washed three times in secondary MHC-binding anchor residues into account.
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