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ORIGINAL RESEARCH |
From the Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, the Department of Microbiology and Immunology, and the Division of Gynecologic Oncology, University of Arkansas, Little Rock, Arkansas; and the Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
Address reprint requests to: Alessandro D. Santin, MD University of Arkansas UAMS Medical Center Division of Gynecologic Oncology 4301 West Markham Little Rock, AR 72205-7199
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Abstract |
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Methods: CD8+ T lymphocytes derived from peripheral blood mononuclear cells stimulated in vitro with autologous ovarian tumor peptide-pulsed dendritic cells were tested for their ability to induce an HLA class I–restricted cytotoxic T lymphocyte response against autologous tumor cells. To correlate cytotoxic activity by cytotoxic T lymphocytes with T cell phenotype, we used two-color flow cytometric analysis of surface markers and intracellular cytokine expression (interferon-
versus interleukin-4).
Results: CD8+ cytotoxic T lymphocyte responses against autologous ovarian tumor cells were elicited in three consecutive women who had advanced ovarian cancer. Although cytotoxic T lymphocyte populations from all women expressed strong cytolytic activity against autologous tumor cells, they did not lyse autologous lymphoblasts or Epstein-Barr virus-transformed cell lines, and they showed negligible cytotoxicity against the natural killer-sensitive cell line K-562. Cytotoxicity against the autologous tumor cells was significantly inhibited by anti-HLA class I (W6/32) and anti-HLA-A2 (BB7-2) monoclonal antibodies. CD8+ cytotoxic T lymphocytes expressed variable levels of CD56 and preferentially expressed interferon- rather than interleukin-4.
Conclusions: Peptide-pulsed dendritic cells induced specific CD8+ cytotoxic T lymphocytes that killed autologous tumor cells from women with advanced ovarian cancer. This finding might contribute to the development of active or adoptive immunotherapy for residual or resistant ovarian cancer after standard surgery and cytotoxic treatment.
Because of the insidious onset and progression of ovarian cancer, about two thirds of women have advanced disease at diagnosis. Although many women with disseminated tumors respond initially to standard combinations of surgery and cytotoxic therapy, nearly 90% of tumors recur and women inevitably die of their disease.1 Thus, novel treatment strategies are needed greatly.
Recent advances in our knowledge of tumor-associated antigens in several human malignancies and insight into mechanisms involved in immune-mediated recognition of them have provided a strong basis for using the immune system as a therapeutic tool for treating cancer. However, with the partial exception of melanoma, immunization using defined antigens is limited for most of human tumors.2 An alternative effective strategy for vaccinating women with tumors might be unfractionated tumor-derived antigens such as whole tumor cells, peptides, or proteins isolated from tumor cells. Effective tumor immunity in several murine tumor models has been induced using professional antigen-presenting cells, such as dendritic cells pulsed with unfractionated tumor-derived antigens.3–5
Unlike murine tumor models, in which the availability of tumor-derived antigens is usually not limited, stable long-term cultures cannot be established for most human tumor explants, including ovarian carcinomas. That limitation might preclude collection of a sufficient amount of tumor antigen for vaccination. Recently, the rapid isolation of class I–presented peptides from viable cells by mild acid elution was described.4,6 In those studies, multiple cycles of acid elution of peptides from tumor cells while preserving cell viability was effective for dendritic cell-based immunization strategies in murine tumor models.4,6 Acid elution of peptides might yield a superior immunogenic source of dendritic cell–presented epitopes than peptides extracted from tumor cells by repeated cycles of freeze-thaw lysis.4
Studies by several groups, including us, recently established the key effect of dendritic cells in the immune system and provided a rationale for using these cells as a natural adjuvant for tumor immunotherapy.7–9 Dendritic cells are the most effective antigen-presenting cells for activating naive T cells,7 and recently the combination of granulocyte-macrophage-colony stimulating factor and interleukin-4 has been used to generate large numbers of dendritic cells for induction of human T cell responses.10 In this study, we used autologous dendritic cells pulsed with acid-eluted peptides from autologous ovarian tumor cells to induce tumor-specific cytotoxic T cell responses in women with advanced ovarian cancer.
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Materials and Methods |
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The natural killer–sensitive target K562 (a human erythroleukemia cell line) was purchased from American Type Culture Collection (Rockville, MD) and maintained at 37C, 5% carbon dioxide (CO2) in complete medium containing RPMI 1640 (Gibco-BRL, Grand Island, NY) and 10% fetal bovine serum (Gemini Bio-products, Calabasas, CA). Fresh ovarian tumor cells were collected from surgical specimens. Single-cell suspensions were collected by processing solid tumor samples under sterile conditions at room temperature as described.11 Viable tumor tissue was minced mechanically in RPMI 1640 to portions no larger than 1–3 mm3 and washed twice with RPMI 1640. The portions of minced tumor were then placed in 250-mL trypsinizing flasks containing 30 mL of enzyme solution (0.14% collagenase Type I [Sigma, St. Louis, MO] and 0.01% DNAse [Sigma, 2000 KU/mg]) in RPMI 1640 and were incubated on a magnetic stirring apparatus overnight at 4C. Enzymatically dissociated tumor was filtered through a 150-µm nylon mesh to generate a single-cell suspension. The resultant cell suspension was washed twice in RPMI 1640 plus 10% autologous plasma. Fresh tumor cell lines were maintained initially in RPMI 1640, supplemented with 15% autologous ascites fluid at 37C, 5% CO2. All cytotoxicity experiments were done with fresh or cryopreserved tumor cultures that had at least 90% viability and contained more than 99% tumor cells.
HLA class I typing of purified CD8+ T cells was done by standard lymphocytotoxicity12 in the tissue-typing laboratory of the bone marrow transplantation and blood transfusion service at the University of Arkansas for Medical Sciences. The ovarian cancer patients had the following haplotypes: HLA A24, A30, B7, B8, BW6, and CW7; HLA A2, A29, B51, B44, BW4, and CW1; and HLA A23, A33, B54, B53, BW4, BW6, CW4, and CW17.
Peptides were extracted from the surface of 5 to 10 x 106 autologous tumor cells either pelleted in a 15-mL polypropylene tube (Falcon, Oxnard, CA) (fresh tumor cells in single-cell suspension) or adherent to flasks (T75 tissue culture flask, Corning Co., Corning, NY), (adherent fresh tumor cultures) as described.6 Adherent cells cultured in vitro in RPMI 1640 plus 15% autologous ascites were washed twice with phosphate-buffered saline (pH 7.4, Gibco-BRL), followed by 5 mL of citrate-phosphate buffer (0.131 M citric acid, 0.066 M NA2HPO4, 0.15 M NaCl, pH 3.0) per T75 flask. Flasks were rocked and suspension cells were mixed by gentle pipetting for 5 minutes at room temperature. For tumor cells in single suspension, 5 mL of citrate-phosphate buffer was added directly to pelleted cells in 15-mL conical tubes for 5 minutes at room temperature. Fresh tumor cells were thereafter salvaged from acid treatment by rapid neutralization of cell pellets or flask-adherent cells with a 10-mL wash with RPMI 1640 containing either 15% autologous ascites or 15% autologous plasma. Tumor cells were then seeded in tissue culture flasks or analyzed by flow cytometry for HLA class I expression as described below. Peptide eluates from cells were centrifuged at 500 g for 3 minutes and further clarified by centrifugation at 1800 g for 5 minutes at 4C. The peptides were stored at -70C in citrate-phosphate buffer until further processing or were processed immediately on Sep-Pak C18 cartridges (Waters, Bedford, MA). Cartridges were attached to 5-mL syringes and were equilibrated with 3 mL of acetonitrile followed by 3 mL of deionized water. The acid eluate was allowed to flow through the column by gravity. The column was washed with 5 mL of deionized water, and material bound to the column was eluted with 1 to 2 mL of 60% acetonitrile/40% deionized water. The eluates were lyophilized in a Speed-Vac (Savant Inc., Farmingdale, NY) and reconstituted in AIM-V medium (Gibco-BRL). Peptides were filter sterilized and stored at -80C until use.
Peripheral blood mononuclear cells were separated from heparinized venous blood by Ficoll-Hypaque (Sigma) density gradient centrifugation and either cryopre-served in RPMI 1640 plus 10% dimethyl sulfoxide (DMSO) and 30% autologous plasma or used immediately for dendritic cell generation. Peripheral blood mononuclear cells collected from 42 mL of peripheral blood were placed in six-well culture plates (Costar, Cambridge, MA) in AIM-V at 0.5 to 1 x 107 per 3-mL per well. After 2 hours at 37C, nonadherent cells were removed, and the adherent cells were cultured at 37C in a humidified 5% CO2/95% air incubator, in AIM-V medium supplemented with recombinant human granulocyte-macrophage-colony stimulating factor (800 U/mL; Immunex, Seattle, WA) and interleukin-4 (1000 U/mL; Genzyme, Cambridge, MA).10 In early experiments, only granulocyte-macrophage-colony stimulating factor (800 U/mL) was used. Every 2 days, 1 mL of spent medium was replaced by 1.5 mL of fresh medium containing 1600 U/mL granulocyte-macrophage-colony stimulating factor and 1000 U/mL interleukin-4, to yield final concentrations of 800 U/mL and 500 U/mL, respectively.10 After 6 or 7 days of culturing, dendritic cells were harvested for pulsing with acid-eluted peptides as described.
After culture, dendritic cells were washed twice in AIM-V and added to 50-mL polypropylene tubes (Falcon). Previous reports showed higher levels of cytotoxic T lymphocyte stimulation when dendritic cells were pulsed with peptides incorporated in liposomes,13 so the cationic lipid N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) (Boehringer Mannheim, Indianapolis, IN) was used to deliver the acid-eluted peptides to dendritic cells. Five hundred microliters of eluted peptides derived from 5 to 10 x 106 tumor cells in AIM-V were mixed with DOTAP (125 µg in 500 µL of AIM-V) in 12 x 75-mm polystyrene tubes at room temperature for 20 minutes. The complex was added to the dendritic cells in a total volume of 2–3 mL of AIM-V medium and incubated at 37C with occasional agitation for 3 hours. The cells were washed twice with phosphate-buffered saline and resuspended in AIM-V as described.
Fresh or cryopreserved responder peripheral blood mononuclear cells were washed and resuspended in AIM-V at 10 to 20 x 106 cells per well in six-well culture plates with tumor peptide-pulsed autologous dendritic cells (ratios from 20:1 to 30:1 responder peripheral blood mononuclear cells:dendritic cells). The cultures were supplemented with recombinant human granulocyte-macrophage-colony stimulating factor (500 U/mL) and recombinant human interleukin-2 (10 U/mL Aldesleukin; Chiron Therapeutics, Emeryville, CA) and incubated at 37C. Recombinant human interleukin-2 (10 U/mL) was added to the cultures thereafter every 3 to 4 days. At day 21, CD8+ T cells were separated from the bulk cultures by positive selection with CD8-Dynabeads (Dynal Inc., Lake Success, NY) and further expanded for 7–10 days using autologous or allogeneic irradiated peripheral blood lymphocytes (5000 cGy) (1 x 106 cells per well) and anti-CD3 monoclonal antibody (0.2 µg/mL) (Ortho Pharmaceutical Corp., Raritan, NJ) in AIM-V plus 5% autologous plasma in 24-well plates (Costar) before being assayed for cytotoxic T lymphocyte activity.
A 6-hour chromium (51Cr) release assay was done as described14 to measure the cytotoxicity of dendritic cell–tumor peptide-stimulated CD8+ T lymphocytes. The K562 tumor cell line was used as a target for the detection of natural killer cell activity. As negative control targets, autologous lymphoblasts were prepared by 3-day stimulation with Con-A (1 µg/mL; Gibco-BRL) in RPMI-1640 plus interleukin-2 (25 U/mL), and Epstein-Barr virus–transformed autologous lymphoblastoid B cell lines were established by coculture of peripheral blood mononuclear cells with Epstein-Barr virus–containing supernatant from the B95.8 cell line in the presence of 1 µg/mL cyclosporine (Sandoz, Camberley, United Kingdom) and were maintained in AIM-V supplemented with 10% human AB serum (Gemini Bioproducts). To determine HLA class I restriction of lysis, monoclonal antibodies were used to block cytotoxicity. The 51Cr-labeled tumor targets were preincubated with monoclonal antibodies specific for monomorphic HLA class I W6/32 or anti-HLA-A2 (BB7-2) (50 µg/mL) or isotype control (50 µg/mL). To determine other molecules on the effector cells involved in lysis, we used monoclonal antibodies against CD3 (10 µg/mL), CD11a/LFA-1 (10 µg/mL), and its isotype control (IgG1
monoclonal antibody isotype standard anti-TNP [10 µg/mL]) (PharMingen, San Diego, CA) to block cytotoxicity in some experiments. The effector cells and 51Cr-labeled targets were then incubated in a final volume of 200 µL/microwell at 37C with 6% CO2.
HLA class I expression on autologous ovarian cancer cells before and after acid elution treatment was monitored with the W6/32 anti-class I monoclonal antibody that recognizes a monomorphic determinant on assembled HLA class I molecules and the HC-10 monoclonal antibody, which binds to dissociated heavy chains6 (kindly provided by Dr M. Crew, University of Arkansas for Medical Sciences, Little Rock, AR). Single-cell suspensions of acid-treated or untreated tumor cells were washed once in RPMI 1640 containing either 15% autologous ascites or 15% autologous plasma, counted, and distributed into 12 x 75-mm tubes at 5 x 105 cells per tube. W6/32 and HC-10 monoclonal antibodies at 1:500 dilution of ascites in phosphate-buffered saline (pH 7.2) supplemented with 0.1% fetal bovine serum were added in a 50-µL volume. A mouse immunoglobulin (Ig)G preparation (IgG2a; Becton Dickinson, San Jose, CA) was used as a negative control. Secondary fluorescein isothiocyanate-labeled goat anti-mouse IgG was purchased from Organon Teknika (Durham, NC). Acid-eluted cells and untreated cells were analyzed by FACScan (Becton Dickinson) using Cell Quest software (Becton Dickinson).
Enriched cultures of CD8+ T cells were phenotyped at the time of cytotoxicity assays to correlate cytolytic specificity with a particular lymphoid subset. Flow cytometry was done using fluorochrome-conjugated monoclonal antibodies directed against human leukocyte antigens Leu-4 (CD3, pan T cells), Leu-3 (CD4, T helper/inducer), Leu-2a (CD8, T cytotoxic/suppressor), Leu-19 (CD56, NK/K cells), Tac (CD25, the IL-2R), anti-HLA-DR (L-243), anti TcR-
/ß, or TcR-/
(Becton Dickinson).
Flow cytometric analysis of intracellular cytokine expression was done using the protocol described by Openshaw et al15 and conducted essentially as described.9 CD8+ T cells were tested at about 6 weeks after priming, after resting for 14 days after the last antigen stimulation. T cells (7.5 x 105/mL) were incubated at 37C for 6 hours in AIM-V 5% autologous plasma plus 50 ng/mL phorbol myristate acetate and 500 ng/mL ionomycin. Then 10 µg/mL Brefeldin A was added for the final 3 hours of incubation. Controls (nonactivated cultures) were incubated in the presence of Brefeldin A only. The cells were harvested, washed, and fixed with 2% paraformaldehyde in phosphate-buffered saline for 20 minutes at room temperature, after which they were washed and stored overnight in phosphate-buffered saline at 4C. For intracellular staining, cells were washed and permeabilized by incubation in phosphate-buffered saline plus 1% bovine serum albumin and 0.5% saponin (S-7900, Sigma) for 10 minutes at room temperature. Activated and control cells were stained with FITC-anti-interferon-, and PE-anti-interleukin-4, and isotype-matched controls (FITC-anti-Ig2a and PE-anti-Ig1) from Becton-Dickinson. After staining, cells were washed twice with phosphate-buffered saline plus 1% bovine serum albumin and 0.5% saponin, once with phosphate-buffered saline plus 0.5% bovine serum albumin, and fixed a second time with 2% paraformaldehyde in phosphate-buffered saline. Analysis was conducted with a FACScan, using Cell Quest software (Becton Dickinson).
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, as in our previous findings,11 HLA class I molecules were highly expressed on fresh ovarian tumor cells. In all fresh tumors evaluated, acid treatment resulted in a drastic reduction of the expression of major histocompatibility complex class I antigens as recognized by monomorphic anti-class I monoclonal antibody W6/32 compared with untreated control cells. In contrast, reactivity with HC-10 anti-heavy chain monoclonal antibody significantly increased after acid treatment, which indicates that class I heavy chains remain associated with cell surface on fresh ovarian cancer cells in pH 3.3 buffer, but ß2m is lost with the bound peptide into the cell free supernatant.6
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represent the mean of not less than five assays for each patient. Cytotoxicity against autologous tumor-cell targets was found for each patient, ranging from 23–52% in the first woman, 18–49% in the second, and 31–53% in the third, at 20 effectors per target. Cytotoxicity against the natural killer–sensitive cell line K562 was detectable only at a low level. The absence of cytotoxicity against autologous Con-A lymphoblasts (data not shown) or autologous Epstein-Barr virus–transformed lymphoblastoid cell lines (Figure 2) showed that, although those cells were highly cytotoxic against autologous tumor cells, they did not kill normal cells or autologous cells infected with Epstein-Barr virus. Blocking studies showed in all cases that the tumor-specific lytic activity against autologous tumor targets was inhibited by pretreatment of tumor targets with monoclonal antibodies specific for HLA class I molecules (W6/32) (range of inhibition 44–72% in the first woman, 50–65% in the second, and 47–74% in the third woman), (Figure 2) but not with pretreatment by isotype controls (data not shown). BB7-2 monoclonal antibody specific for HLA-A2 also blocked tumor cell killing in the second woman (range of inhibition 44–56%). Those data indicate the importance of HLA-A2 in presenting immunogenic epitope(s) by ovarian tumor cells. In the first and third women we also showed that monoclonal antibodies specific for anti-CD11a (lymphocyte functional antigen-1) also blocked tumor lysis, with the range of inhibition 32–60% in the first woman and 44–64% in the third. Those findings suggested that the CD11a-CD54 adhesion pathway has a critical effect on CD8+ T cell-mediated lysis of ovarian tumor target cells (Figure 2). Anti-CD3 monoclonal antibody failed to block cytotoxicity (data not shown).
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). Different percentages of CD8+ T lymphocytes (range 8–30%) coexpressed the CD56 surface antigen during culture. However, CD56 coexpression on CD8+ T cells, previously reported during the characterization of cytotoxic T lymphocyte populations from patients with cervical cancer,9 did not appear to be a stable phenotype. Several experiments found that expression of that marker was lost by most of the previously positive CD8+/CD56+ T cells when the cells were cultured in low doses of interleukin-2, only to be re-expressed after restimulation.
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+/interleukin-4- and interferon--/-interleukin-4+ subsets we used recently developed flow cytometric techniques to detect intracellular cytokine expression at the single cell level. Two-color flow cytometric analysis of intracellular interferon- and interleukin-4 expression by CD8+ cytotoxic T lymphocytes was done after at least 6 weeks of culture. As shown in Figure 4, most CD8+ T cells from each woman expressed intracellular interferon-, whereas a second subset expressed only interleukin-4, and a third subset expressed both cytokines. Similar results were seen in several repetitive analyses from different cytotoxic T lymphocyte primings for each woman. Unactivated (ie, resting) CD8+ T cells from all three women did not stain for interferon- or interleukin-4 (data not shown).
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Although the presence and distribution of antigenic peptides and cytotoxic T lymphocyte epitopes presented by freshly isolated epithelial ovarian tumors is still poorly known, recent studies have shown unequivocally that there are multiple tumor antigens that can be recognized by cytotoxic T lymphocytes and used as target molecules to induce autologous tumor cell lysis in vitro.21–24 However, freshly isolated ovarian tumors are difficult to grow, and their establishment in long-term culture to achieve the necessary cell number for vaccination purposes is possible only for a few patients. Acid elution of peptides (which does not result in significant cell lysis) has been reported in murine models to be a superior source of dendritic cell-presented epitopes than peptides extracted from tumor cells by repeated cycles of freeze-thaw lysis.4
In this study, we found that autologous dendritic cells pulsed with acid-eluted peptides derived from freshly isolated autologous ovarian tumor cells stimulated a specific CD8+ cytotoxic T cell response that can kill tumor cells from women with advanced stage ovarian cancer. In that regard, the numbers of tumor cells we used to produce ovarian tumor acid-eluted peptides for dendritic cell pulsing were much lower (0.5 to 1 x 107 tumor cell equivalent) than those reported in similar studies using murine tumor models (eg, 1 x 108 to 1 x 109 tumor cell equivalent).4 Those data suggest that relatively few fresh tumor cells might be sufficient to provide the threshold of immunogenic peptides necessary for efficient in vitro stimulation of ovarian tumor-specific cytotoxic T lymphocytes in women with ovarian cancer. We found that a proportion of the autologous tumor-specific cytotoxicity was inhibited by anti-HLA class I antibodies, which indicates that most cytotoxicity against autologous tumor cells was mediated by antigen-specific HLA class I-restricted cytotoxic T lymphocytes. In the second woman blocking studies using monoclonal antibodies specific for HLA-A2 class I molecules showed a reduction of autologous tumor cell killing. Those observations agree with previous reports24,25 and further support the importance of HLA-A2 class I molecules in the presentation of immunogenic peptides by ovarian cancer cells. We also found that monoclonal antibodies specific for CD11a, but not CD3, were able to block tumor lysis, which suggests that the CD11a-CD54 adhesion pathway might have a critical influence on effective CD8+ T cell mediated lysis of ovarian tumor target cells. Killing of K562 cells was low for all patients, and autologous lymphoblastoid cell lines or ConA-activated lymphoblasts were also killed at only a low level by tumor-specific cytotoxic T lymphocytes, confirming that although these cytotoxic T lymphocytes were highly cytolytic for autologous tumor cells, they did not respond against autologous normal cells. However, our results did not exclude the possibility that the cytotoxic T lymphocytes recognize tissue-specific rather than tumor-specific antigens. For ovarian cancer, that would not be a limitation to immunotherapy because first-line treatment is surgery, including total abdominal hysterectomy and bilateral salpingo-oophorectomy, so patients lack normal ovarian tissue.
Pure populations of CD8+ T cells from all subjects showed a variable but significant CD8+/CD56+ subpopulation. Although CD56 can be regarded as a natural killer lineage marker, the cytotoxic T lymphocytes described in this study were HLA class I restricted. Those results agreed with our previous findings in women with cervical cancer.9 We suggest that CD56 expression on tumor-specific CD8+ cytotoxic T lymphocytes generated by tumor antigen–pulsed dendritic cells against different human tumors can be regarded as an activation antigen associated with cytotoxic function, rather than a lineage-specific marker.
Several studies documented the deficiency of antitumor effector mechanisms in women with advanced stages of ovarian cancer.25,26 Recently a significant dysfunction of type 1 T cell responses in tumor-bearing hosts was reported,27,28 suggesting that tumor progression might be associated with a preferential type 2 T cell response. T cell-mediated protection from viral infection and control of tumors is believed to be promoted by type 1 cytokine responses and impaired by type 2 cytokine responses.29 In general, type 1 T cells (CD4 or CD8) express interleukin-2, interferon-, and tumor necrosis factor-/ß, and are cytotoxic, whereas type 2 T cells express interleukin-4, interleukin-5, interleukin-6, interleukin-10, and interleukin-13, provide efficient help for B cell activation, and are noncytotoxic. In this study, two-color flow cytometric analysis of intracellular interferon- and interleukin-4 suggested that tumor peptide-pulsed dendritic cells stimulated CD8+ T cells from ovarian cancer patients showed, even at this advanced stage of the disease, a probably type 1 bias in cytokine expression. Those data suggest that dendritic cell–based antigen presentation can, at least in vitro, induce differentiation of type 1 CD8+ T cells that secrete interferon- and are endowed with significant cytotoxic activity against autologous tumor cells. Taken together the findings suggest that appropriate in vitro immunization might induce an efficient T cell immunity against ovarian tumor antigens even after lengthy tumor burden.
The findings of this study show the potential feasibility of either tumor peptide-pulsed–dendritic cell vaccines or adoptive immunotherapy with tumor antigen-pulsed, dendritic cell–primed T cells as strategies for treating residual disease after standard surgical cytotoxic treatment for ovarian cancer. The design and implementation of clinical trials will ultimately determine its validity.
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Footnotes |
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Received February 2, 2000. Received in revised form March 15, 2000. Accepted April 13, 2000.
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