[CANCER RESEARCH 61, 5145–5152, July 1, 2001] Somatically Mutated Regions of Immunoglobulin on Human B-Cell Lymphomas Code for Peptides That Bind to Autologous Major Histocompatibility Complex Class I, Providing a Potential Target for Cytotoxic T Cells1 Clair S. Gricks, Eira Rawlings, Letizia Foroni, J. Alejandro Madrigal, and Peter L. Amlot2 Departments of Immunology [C. S. G., E. R., P. L. A.] and Haematology [L. F., J. A. M.], Royal Free and University College Medical School, London NW3 2PF, United Kingdom ABSTRACT (10). Alternatively, immunization with idiotypic proteins or DNA vaccines without adjuvant produced low levels of idiotypic antibody Lymphoma-derived immunoglobulin idiotype (Id) is a well-character- and poor protective immunity in mice and humans (10, 11). Addition ized, tumor-specific antigen on B-cell malignancies. Immunotherapy using lymphoma immunoglobulin can lead to clinical responses mostly associ- of an adjuvant, for example tetanus toxin fragment C, to DNA ated with anti-Id antibody. We cloned the Id from B-cell lymphomas, encoding the idiotype induced higher levels of idiotypic antibodies in sequenced them, and used bioinformatics to select autologous MHC class mice (12), and this approach is currently being tested in humans. I binding peptides from somatically mutated regions of the lymphoma Id. Although the vast majority of studies have used anti-idiotype antibody Peptides from patients who were HLA-A1, HLA-A2, HLA-A3, or HLA- as an indicator of immune response, more recent work has focused on A11 positive were analyzed in the T2 stabilization assay and a competitive the generation of specific cellular immunity. Immunization of follic- peptide-binding assay. By both methods, approximately half of the pep- ular lymphoma patients with idiotypic protein plus granulocyte/ tides analyzed, regardless of HLA type, bound with intermediate or high macrophage-colony stimulating factor, induced secretion of tumor affinity. Peptide binding affinity was similar to viral peptide sequences known to provide targets for cytotoxic T cells. Further investigation of necrosis factor from postvaccine PBMCs, shown to be mediated by ϩ ϩ lymphocyte responses to stimulation by autologous Id peptides versus Id CD4 and CD8 T cells (13). Specific lysis of autologous tumor cells peptides from other patients revealed that three of five patients in com- was also observed and was blocked by antibodies against MHC class plete remission or with low volume, stable disease responded to self- I, indicating the role of CD8ϩ cytotoxic T cells. Immunization of peptides by IFN-␥ secretion greater than that seen with non-self peptides, lymphoma patients with idiotype protein-pulsed autologous dendritic whereas none of five patients with progressive disease responded to their cells induced cellular proliferative responses rather than humoral own lymphoma Id. We have shown that mutated regions of lymphoma Id immunity (14). A large body of evidence favors cell-mediated rather contain MHC class I binding peptides that are potential targets for cytotoxic T cells. Immunotherapy using the tumor-specific mutated re- than antibody control of tumors, and this is supported by studies in gions from lymphoma Id avoids the need to break innate tolerance toward melanoma (15, 16) and virally induced tumors (17). the germ-line protein sequences present on normal and malignant B cells. The increased knowledge of epitopes within tumor-associated pro- teins recognized by CTL and bioinformatics enabling prediction of peptide binding to HLA class I have provided tools with which to INTRODUCTION search for the existence of CTLs and immunogenic peptides within Immunotherapy of human cancers requires suitably immunogenic, tumors from patients with malignancy (16, 18, 19). This approach has tumor-specific or -associated antigens. Surface immunoglobulin pro- also been used to monitor CTL responses in patients infected with vides a tumor-specific target in patients with B-cell malignancies viruses such as cytomegalovirus and EBV (20, 21). A recent study by because of its unique method of synthesis (1). The random insertion Trojan et al. (22) described HLA-A2-restricted peptide epitopes from of nucleotides between V(D)J gene segments during antigen-indepen- the tumor-derived immunoglobulin variable region from patients dent rearrangement of immunoglobulin genes followed by somatic mainly with CLL. Some of the peptides were shared among patients, hypermutation, occurring in germinal centers as a result of antigen indicating their unmutated, germ-line nature. The authors described stimulation, leads to immunoglobulin sequences not coded in the the in vitro generation of donor and some patient CTLs against the germ-line DNA (2, 3). The unique variable region of rearranged peptides and reported low-level killing of CLL tumor cells. immunoglobulin is known as its Id.3 Follicular lymphomas and my- The aims of our study were to define peptide sequences from elomas have a high rate of somatic hypermutation (4–7), which somatically mutated regions in lymphoma immunoglobulin Ids capa- increases the probability that their Ids will be immunogenic. ble of binding to the patient’s own HLA class I molecules and Immunization with idiotypic protein and adjuvant induces anti- hypothetically with a greater immunogenicity, because the mutated idiotypic antibodies in both mice (8) and humans in the absence of regions should not be regarded as self. The mutant cell line, T2, clinically detectable lymphoma (9), indicating the immunogenic po- provided a means of testing peptide binding to the HLA-A2 allele by tential of Ids. The production of anti-idiotypic antibodies has been the ability to up-regulate and stabilize cell surface expression of shown to mediate protection against tumor challenge in mouse models HLA-A2 peptide complexes (23). In addition, peptides of lymphoma Ids from patients with HLA-A3 were tested on a T2 cell line trans- Received 1/21/01; accepted 5/1/01. fected with HLA A3 (24). Selected peptides restricted by A1, A2, A3, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with and A11 were also tested for binding in a competitive inhibition assay 18 U.S.C. Section 1734 solely to indicate this fact. using either LCLs derived from the patients themselves or LCLs 1 Supported in part by the Leukaemia Research Fund (to E. R.) and by the Marc Fisher homozygous for the HLA type of interest. Finally, stimulation of Trust (to C. S. G.). 2 To whom requests for reprints should be addressed, at Department of Immunology, patient lymphocytes by their own idiotypic peptides was examined in Royal Free and University College Medical School, Rowland Hill Street, London NW3 ELISPOT assays. The demonstration of frequent self-HLA binding 2PF. Phone: 44-20-7472-6266; Fax: 44-20-7431-3416; E-mail: [email protected]. 3 The abbreviations used are: Id, idiotype; PBMC, peripheral blood mononuclear cell; lymphoma idiotypic peptides and lymphocytes from lymphoma pa- CLL, chronic lymphocytic leukemia; LCL, lymphoblastoid cell line; ELISPOT, enzyme- tients in long-term remission capable of reacting to idiotypic peptides linked immunospot; RT-PCR, reverse transcription-PCR; MFI, mean fluorescence inten- ␥ sity; FR, framework region; CDR, complementarity-determining region; DLBL, diffuse by IFN- secretion suggests that mutated idiotypic peptides provide a large B-cell lymphoma; FCL, follicle center lymphoma. target for control of lymphomas by cell-mediated immune responses. 5145 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2001 American Association for Cancer Research. LYMPHOMA IDIOTYPIC PEPTIDE BINDING TO MHC CLASS I MATERIALS AND METHODS A2, A3, or A11 (cell lines JY, HOM-1, VAVY, and FB) or the patient’s own B-LCL were washed twice in PBS and left to rest as a pellet on ice for 5 min. Patients. Lymphoma patients were classified according to the REAL clas- The cells were then acid stripped for 90 s using ice-cold Na2HPO4 buffer, pH sification (25). Lymphoid tissue was analyzed from 17 patients with diffuse 3.1 for A1, pH 3.2 for A2, and pH 2.9 for A3 and A11. The cells were buffered large cell lymphoma, 16 with follicle center lymphoma, 6 with B-cell small with ice-cold Iscove’s modified Dulbecco’s medium (ICN Flow, Basingstoke, lymphocytic lymphoma, 2 with mantle cell lymphoma, 2 with marginal zone Hampshire, United Kingdom), and then washed and resuspended at 1 ϫ 106/ml lymphoma, 1 with plasma cell myeloma, and 1 with lymphoplasmacytoid ␮ ␤ in Iscove’s modified Dulbecco’s medium plus 7.5 g/ml 2-microglobulin lymphoma. All patients were referred to our institution and treated according (Cymbus Biotechnology Ltd.). Twenty-five ␮l of fluorescent-labeled reference to United Kingdom standard lymphoma protocol. peptide [HLA-A1, YLEPACFITCAKY; HLA-A2, FLPSDCFITCFPSV; HLA- Genomic DNA Isolation. Diagnostic biopsies from 45 patients with B-cell A3/11, KVFPCFITCALINK (Department of Immunohematology and Blood malignant lymphomas were processed for DNA extraction using Proteinase K Bank, Leiden University Medical Center)] at a final concentration of 133 nM and a Puregene kit (Flowgen, Lichfield, Staffordshire, United Kingdom). were incubated with 25 ␮l of Id competitor peptide (MWG-Biotech) at final Patients were assessed for IgH clonality by Southern blotting and hybridization concentrations of 0.5–33.3 ␮M in a 96-well, U-bottomed plate with 100 ␮l to a JH probe, as described previously (26, 27). Patients with clonal JH pattern (1 ϫ 105) of cells for 24 h at 4°C (42). The percentage inhibition demonstrated were processed for amplification of the rearranged IgH genes as detailed by immunoglobulin peptides competing with the FITC-labeled reference pep- below. tide was calculated as described (42). PCR. PCR was performed using the manufacturer’s instruction (Promega, ELISPOT Assay for IFN-␥. PBMCs (1 ϫ 105) were plated in triplicate in Southampton, United Kingdom) in a 30-␮l volume containing 1 ␮g of genomic round-bottomed, 96-well plates and stimulated for 18 h with 50 ␮g/ml peptide DNA and 100 pM of each primer.