Chronic Lymphocytic Leukemia-reactive T Cells during Disease Progression and after Autologous Tumor Cell Vaccines 1

Purpose: Tumor-reactive T cells were measured in patients with chronic lymphocytic leukemia (CLL) because vaccines that increase the activity of these cells might lead to better disease control. Experimental Design: Proliferation and ELISPOT assays (for T cells producing IFNafter stimulation by CD40activated CLL cells) were used to determine the prevalence of tumor-reactive T cells in 25 CLL patients at various stages of disease progression. The effects of vaccines, composed of autologous-oxidized tumor cells, on both the clinical course and tumor-reactive T-cell numbers were then determined in 2 patients. Results: CLL-reactive T cells were found at frequencies of >10 3 in 6 of 11 patients. Significant proliferation was found in 15 of 25 patients and correlated with clinical stage. The inability to measure CLL-reactive T cells in the remaining patients was not uniformly a result of generalized T-cell dysfunction or defective antigen presentation by CD40-activated CLL cells. CLL-reactive T-cell frequencies increased in response to vaccination with oxidized autologous tumor cells in a patient with preexisting CLL-reactive T cells but not in a patient where tumor-reactive T cells were undetectable in the ELISPOT assay. Conclusions: Tumor-reactive T cells exist in some CLL patients (mainly during earlier stages of disease) and may potentially mediate therapeutic responses if their numbers and activation states can be sufficiently increased by tumor vaccines. INTRODUCTION CLL is incurable with conventional chemotherapy (1). However, its origin in transformed anergic self-reactive CD5 B cells (2) that are subject to T-cell regulation (3) suggests that immunotherapeutic approaches may lead to better disease control. Other observations that support the use of immunotherapy in CLL include (a) the physical association of CLL and T cells in secondary lymphoid organs, which assures interactions between tumor-reactive T cells and tumor cells (4); (b) spontaneous remissions associated with heightened immune activity because of viral infections (5); (c) clinical responses to immunomodulatory cytokines (6); and (d) long-term disease-free survival after allogeneic bone marrow transplantation (7), possibly from a T-cell-mediated graft-versusleukemia effect (8). Immune control of tumors, including CLL, is believed to be mediated mainly by CTLs that recognize tumor antigens (9, 10). Cytotoxic CD8 T cells are mainly responsible for the destruction of epithelial tumors (11), but both CD4 and CD8 CTLs may kill B-cell tumors (3, 12). The purpose of cancer vaccines is to increase the number of these tumor-reactive CTLs and maintain their activity for long enough to clear tumor cells. In theory, cancer vaccines should both promote tumor clearance and prevent relapse by providing long-term antitumor immunity. Unfortunately, tumor vaccines, although occasionally yielding dramatic results, have not generally achieved their objective (10). Our laboratory has been evaluating the requirements for successful vaccination of CLL patients because of the need for new treatments, rationale for vaccines in this disease, and as a general model for immunologically susceptible tumors. At a minimum, successful cancer vaccines require tumor antigens that can be presented with appropriate costimulatory signals to T cells able to respond to these antigens. With the possible exception of the immunoglobulin idiotype (13), the antigens recognized by CLL-reactive T cells are generally unknown. By analogy with other tumor associated antigens (14), CLL antigens are likely encoded by genes that are mutated or overexpressed during oncogenesis and may be unique to each patient. Problems posed by the unknown character and uniqueness of CLL antigens may be addressed by using vaccines based on the patient’s own tumor cells (15). Successful cancer vaccines also require T cells that can respond to them. This requirement may not always be satisfied Received 9/16/02; revised 11/20/02; accepted 11/25/02. 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 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from the Leukemia Research Fund of Canada (to D. S.), Canadian Institutes of Health Research (to D. S., N. B.), and from Vasogen, Inc. (to D. S.). D . S. is a physician-scientist supported by Cancer Care Ontario. 2 To whom requests for reprints should be addressed, at Division of Molecular and Cellular Biology, Research Institute, S-116A, Research Building, Sunnybrook and Women’s College Health Sciences Center, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada. Phone: (416) 480-6100, ext. 2510; Fax: (416) 480-5737; E-mail: spanerd@ srcl.sunnybrook. utoronto.ca. 3 The abbreviations used are: CLL, chronic lymphocytic leukemia; HI, heat inactivated; APC, antigen-presenting cell; t-CD40L, CD40-CLL, CD40L-activated CLL cells; PMA, phorbol 12-myristate 13-acetate; PE, phycoerythrin; MFI, mean fluorescence intensity. 1656 Vol. 9, 1656–1665, May 2003 Clinical Cancer Research Research. on April 11, 2017. © 2003 American Association for Cancer clincancerres.aacrjournals. org Downloaded from for a number of reasons. Tumor antigen-reactive T cells may never have been present [because many tumor antigens are actually self-antigens (16)], they may have been lost during tumor progression (17), or they may have been suppressed by regulatory T cells (18). Tumor vaccines may be improved by inhibiting suppressor cells but are unlikely to be successful in the absence of tumor-reactive T cells. The studies in this article were carried out to determine whether tumor-reactive T cells could be found in patients at various stages of CLL. ELISPOT (19, 20) and proliferation assays (with autologous-activated CLL cells as stimulators) were used to reveal the presence of tumor-reactive T cells without requiring the precise identification of the antigenic targets. We also determined if CLL-reactive T-cell numbers could be increased in vivo by autologous vaccines composed of oxidized tumor cells. PATIENTS AND METHODS Blood Samples. After obtaining informed consent, heparinized blood (30–40 ml) was taken from 25 CLL patients (Tables 1 and 2). Four blood samples were also obtained from hemochromatosis patients undergoing phlebotomy to prevent iron overload (21). Protocols to obtain these samples were approved by the local Institutional Review Board. Complete blood counts were obtained from the hospital clinical hematology laboratory. Cell Purification. B, CLL, and T cells (and sometimes CD4 and CD8 subsets) were immediately isolated from each blood sample using the RosetteSep-negative selection technique (StemCell Technologies, Vancouver, British Columbia, Canada) according to the manufacturer’s instructions (with minor modifications). Briefly, 75% of the plasma volume was removed from heparinized whole blood samples to concentrate the peripheral blood mononuclear cells, increase the yield of isolated cells, and minimize the required antibodies. The collected plasma was HI at 56°C for 40 min for subsequent experimental use and cryopreservation. Total T cells, or CD4 and CD8 subsets, were obtained with antibodies against CD16, CD19, CD36, CD56, and glycophorin A or against CD16, CD19, CD36, CD56, CD8 (or CD4), and glycophorin A, with 96, 94, and 92% purity, respectively. Peripheral blood B cells were isolated with antibodies against CD2, CD3, CD14, CD16, CD56, and glycophorin A yielding percentages of CD19 , CD19 /CD5 , and CD3 cells of 98, 96, and 0.1%,