Lymphoma Vaccines for Active Immunotherapy of T Cell Are More

Tumor Cell Lysate-Pulsed Dendritic Cells Are More Effective Than TCR Id Protein Vaccines for Active Immunotherapy of T Cell Lymphoma This information is current as of October 3, 2021. Erin Gatza and Craig Y. Okada J Immunol 2002; 169:5227-5235; ; doi: 10.4049/jimmunol.169.9.5227 http://www.jimmunol.org/content/169/9/5227 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Tumor Cell Lysate-Pulsed Dendritic Cells Are More Effective Than TCR Id Protein Vaccines for Active Immunotherapy of T Cell Lymphoma1

Erin Gatza2* and Craig Y. Okada†

TCR Id protein conjugated to keyhole limpet hemocyanin (KLH) (TCR Id:KLH) and injected with a chemical adjuvant (QS-21) induces a protective, Id-specific immune response against the murine T cell lymphoma, C6VL. However, Id-based immunotherapy of C6VL has not demonstrated therapeutic efficacy in tumor-bearing mice. We report here that C6VL lysate-pulsed dendritic cells (C6VL-DC) vaccines display enhanced efficacy in both the prevention and the therapy of T cell lymphoma compared with TCR Id:KLH with QS-21 vaccines. C6VL-DC vaccines stimulated potent tumor-specific immunity that protected mice against lethal challenge with C6VL and significantly enhanced the survival of tumor-bearing mice. Tumor-specific proliferation and secretion of IFN-␥ indicative of a Th1-type immune response were observed upon ex vivo stimulation of vaccine-primed lymph node cells. Downloaded from Adoptive transfer of immune T cell-enriched lymphocytes was sufficient to protect naive recipients from lethal tumor challenge. Furthermore, CD8؉ T cells were absolutely required for tumor protection. Although C6VL-DC and control vaccines stimulated low levels of tumor-specific Ab production in mice, Ab levels did not correlate with the protective ability of the vaccine. Thus, tumor cell lysate-pulsed DC vaccines appear to be an effective approach to generate potent T cell-mediated immune responses against T cell malignancies without requiring identification of tumor-specific Ags or patient-specific Id protein expression. The

Journal of Immunology, 2002, 169: 5227–5235. http://www.jimmunol.org/

cell malignancies are diverse in their composition, aggres- response against a murine T cell lymphoma, C6VL. Vaccination siveness, and response to treatment. Despite modern ther- with recombinant TCR Id protein conjugated to keyhole limpet T apies, including radiation, chemotherapy, and bone mar- hemocyanin (KLH)3 and injected with a chemical adjuvant (TCR row transplants, most patients with T cell cancers remain incurable Id:KLHϩQS-21) stimulated both humoral and cellular anti-Id re- due to disease recurrence (1). Immunotherapeutic approaches to sponses in mice. Despite Id-specific Ab production in vaccine re- treat lymphoma patients once they attain a state of minimal resid- cipients, anti-tumor immunity was primarily mediated by CD8ϩ T ual disease may reduce or delay recurrence and enhance survival cells (10, 11). In subsequent experiments vaccination of mice with (2). To date, efforts to develop immunotherapy for B and T cell adenoviral vectors containing chimeric TCR Id also stimulated by guest on October 3, 2021 lymphomas have largely focused on the use of tumor-derived id- CD8ϩ-mediated anti-tumor responses in the C6VL model (12). iotype protein as a source of defined tumor-specific epitopes. Id This approach did not result in enhanced levels of survival com- proteins expressed on B cell lymphomas have been exploited in pared with vaccination with TCR Id:KLHϩQS-21, however. We numerous murine models (3Ð7) as tumor-specific targets for the hypothesized that combining the potent adjuvant potential of den- development of tumor immunotherapy and have also been associ- dritic cells (DC) in stimulating cell-mediated immunity against ated with enhanced clinical outcome (8, 9). For the development of tumors with the ability to target multiple tumor-specific Ags would immunotherapeutic approaches for T cell lymphoma, the studies allow increased efficacy in the immunotherapy of T cell lymphoma have been far fewer in number, but have also concentrated on compared with TCR Id:KLHϩQS-21 vaccines. using tumor-derived Id epitopes to generate anti-tumor immunity. The use of DC in the development of effective prophylactic and Okada and colleagues (10, 11) demonstrated that vaccination with therapeutic vaccines for a variety of cancers has been the focus of tumor-derived TCR Id protein generated an effective anti-tumor intense investigation by many groups. DC have been targeted in this effort largely due to their unique ability to initiate potent primary and secondary immune responses (13) and to control the type *Graduate Program in Immunology, University of Michigan Medical School, Ann of immune response that is induced (14). DC loaded with tumor- Arbor, MI 48101; and †Division of Hematology and Oncology, University of Mich- derived DNA, RNA, protein(s), or peptides have been used as an igan and Veterans Administration Ann Arbor Health Care Systems, Ann Arbor, MI 48105 efficacious vaccine in multiple murine tumor models (15Ð26). DC have also been effective as cellular adjuvants for B cell Id protein Received for publication June 24, 2002. Accepted for publication September 3, 2002. vaccines. Loading DC with B cell Id:KLH stimulated levels of 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 anti-tumor immunity superior to Id:KLH injected with a chemical with 18 U.S.C. Section 1734 solely to indicate this fact. adjuvant (27). In patients, loading DC with B cell Id protein with 1 This work was supported in part by the Office of Research and Development, Med- ical Research Service, Department of Veterans Affairs, and a G&P Medical Research Award. E.G. was supported by National Institute of Allergy and Infectious Diseases Training Grant T32AI07413 and National Cancer Institute Training Grant 3 Abbreviations used in this paper: KLH, keyhole limpet hemocyanin; CM, complete T32CA88784. medium; DC, dendritic cells; C6VL-DC, C6VL lysate-pulsed DC; MAC, metal af- 2 Address correspondence and reprint requests to Erin Gatza, Graduate Program in finity column; MBL-2-DC, MBL-2 lysate-pulsed DC; MLF, mean log fluorescence; Immunology, University of Michigan, Veterans Administration Ann Arbor Health SI, stimulation index; S:R ratio, stimulator:responder ratio; TCR Id:KLHϩQS-21, Care Systems Research Service 11R, Ann Arbor, MI 48185. E-mail address: TCR Id conjugated to keyhole limpet hemocyanin and mixed with QS-21; VPLN, [email protected] vaccine-primed lymph nodes.

or without carrier protein (KLH) resulted in the generation of Id- CD3, CD4, and H-2Db surface proteins (37). C6VL does not express MHC specific cellular immune responses (9, 28, 29) in addition to the class II or Fas (10). MBL-2 (H-2b) is a T cell lymphoma cell line of largely humoral immune response observed in patients receiving B C57BL/6 origin (provided by I. L. Weissman, Stanford, CA) that was used as a control T cell lymphoma lysate and a control tumor challenge. B16/ cell Id vaccines without DC. F10 (American Type Culture Collection, Manassas, VA) is a melanoma The ability to target multiple tumor Ags may increase the mag- derived from C57BL/6 mice and was used as a non-lymphoid tumor lysate nitude and diversity of anti-tumor responses, thus preventing tu- control. mor from escaping responses limited in repertoire (30). Loading Antibodies DC with proteins from tumor cell lysates may result in the presentation of a broader array of tumor-specific Ags than what may mAb 124-40 (mouse IgG1␬) recognizes a determinant on the C6VL TCR ␣ be presented using Id protein (31), including multiple epitopes for -chain V region (36). mAb 2.43 (Rat IgG2b) recognizes mouse CD8.2. ϩ ϩ mAb SFR8-B6 (rat IgG2b) recognizes human HLA-Bw6. The 2.43 hy- both CD4 and CD8 T cells (32). In addition, tumor cell lysates bridoma was purchased from American Type Culture Collection. The contain agents known to induce maturation of DC (32). Mature DC SFR8-B6 hybridoma was provided by J. Parnes (Stanford, CA). mAb have an enhanced capacity to prime potent Ag-specific CTL ac- 124-40 was purified from culture supernatant over a protein A-Sepharose tivity and stimulate IFN-␥ release by CD4ϩ Th cells. column. 2.43 and SFR8-B6 were purified from serum-free hybridoma culture supernatants by ammonium sulfate precipitation, followed by strong Recently, two groups have demonstrated that tumor lysate-spe- anion exchange chromatography. Ab concentrations were determined by cific cytotoxic T lymphocytes can be generated ex vivo against bicinchoninic acid assay (Pierce, Rockford, IL). FITC-labeled anti-CD4 autologous primary multiple myeloma (33) and B cell chronic (GK1.5, isotype rat IgG2b,␬), anti-CD80 (16-10A1, hamster IgG), anti- b lymphocytic leukemia (34) tumor samples using tumor lysate- CD86 (GL1, rat IgG2a,␬), anti-CD11c (HL3, hamster IgG), anti-I-A ␬ ␬ ⑀ pulsed DC as stimulators. We demonstrate here that tumor lysate- (AF6-120.1, mouse IgG2a, ), anti-CD40 (3/23, rat IgG2a, ), anti-CD3

(145-2C11, hamster IgG), and anti-CD19 (1D3, rat IgG2a,␬); PE-labeled Downloaded from pulsed DC stimulate a potent anti-tumor response against murine T anti-CD8b.2 (53-5.8, rat IgG1,␬); and isotype controls were purchased cell lymphoma in vivo. Vaccination of mice with T cell lympho- from BD Biosciences (San Diego, CA). PE-labeled anti-CD8b.2 (53-5.8) ϩ ma-pulsed DC (C6VL-DC) significantly enhanced the survival of was used to determine CD8 T cell depletion in mice and is non-cross- ϩ ϩ tumor-challenged mice compared with the previously reported blocking with mAb 2.43. FITC-labeled goat anti-mouse IgG IgM (H L) was purchased from Jackson ImmunoResearch Laboratories (West Grove, TCR Id:KLH in QS-21 vaccines. Anti-tumor immunity stimulated PA). Purified and biotinylated forms of rat anti-mouse IFN-␥ and rat anti- by C6VL-DC was lysate specific and required the presence of mouse IL-4 mAbs for ELISA were purchased from Caltag (Burlingame, CD8ϩ T cells. The vaccines stimulated a potent cellular response CA). Streptavidin-HRP was purchased from Pierce (Rockford, IL). http://www.jimmunol.org/ that was characterized ex vivo by tumor-specific proliferation and C6VL TCR Id vaccine preparation IFN-␥ secretion by vaccine-primed lymphocytes. Lastly, C6VL lysate-pulsed DC vaccines significantly enhanced the survival of The C6VL TCR Id protein vaccine was used for comparison with prior tumor-bearing mice. This study is the first to directly compare the experiments. The C6VL TCR Id protein was purified from a recombinant overexpressing cell line. Briefly, DNA constructs of ␣- and ␤-chains of the efficacy of defined protein epitope-based and tumor lysate-pulsed C6VL TCR were modified to contain coding sequences for a thrombin DC-based vaccine approaches for the immunotherapy of lympho- cleavage site, coiled-coil (derived from a leucine zipper protein), and six mas. Our results demonstrate that tumor lysate-pulsed DC are ef- histidine residues. The DNA constructs were cloned into the expression ficacious in stimulating reactivity against T cell lymphoma that is vector, pSR␣SD5 (10) and transfected into BW5147 cells by electropora- tion. A cell line was isolated that expressed ϳ3 mg/L C6VL TCR Id pro- by guest on October 3, 2021 capable of clearing existing tumor as well as preventing future tein in the culture supernatant. The TCR Id protein was purified from the tumor growth. Tumor lysate-pulsed DC vaccines out-perform the culture supernatant on a 124-40 mAb affinity column and then on a metal idiotype-based vaccine strategy currently being pursued for the affinity column (MAC). The protein was eluted from the MAC with imi- immunotherapy of T cell lymphomas. dazole and then digested with thrombin. After cleaving the coiled-coil/His tag from the TCR Id protein with thrombin, the digest was run over a Materials and Methods second MAC, and the TCR Id protein was collected in the flow-through. The TCR Id vaccines were prepared by conjugating the recombinant TCR Mice Id protein to KLH (1:1 ratio, 35 ␮g TCR Id/vaccine) and then mixing with ␮ Female C57BL/6 mice (H-2b) were purchased from The Jackson Labora- QS-21 (10 g/vaccine) for s.c. delivery. tory (Bar Harbor, ME) at 6 wk of age. The animals were housed in the Ann Generation of bone marrow-derived DC Arbor Veteran’s Affairs animal medical unit under specific pathogen-free conditions according to the guidelines drafted in the Animal Component of Bone marrow cells were flushed from the femurs and tibias of C57BL/6 Research Protocol that is implemented in Veterans Affairs research centers. mice and depleted of erythrocytes using Sigma Red Cell Lysis Buffer (8.3

The mice were between 7 and 9 wk of age when vaccine protocols were g/L NH4Cl in 0.01 M Tris-HCl buffer, pH 7.2). The resulting cells were initiated. cultured at a density of 1.0 ϫ 106 cells/ml in either CM (Figs. 1 and 2B) Media and cytokines or AIM-V serum-free medium (all other experiments) containing 10 ng/ml rGM-CSF and rIL-4. On day 3 of culture, fresh cytokines were added, and AIM-V serum-free medium (Invitrogen, Carlsbad, CA) was used to derive the total culture volume was increased by 50% with the addition of fresh DC and to generate DC-based vaccines. DC used for in vitro assays and medium. After 5 days of culture, nonadherent cells were harvested by C6VL for tumor challenges were grown in complete medium (CM), con- gentle pipetting and were suspended in CM or AIM-V to a density of 4.0 ϫ 6 sisting of RPMI 1640 supplemented with 10% FCS, 0.1 mM nonessential 10 cells/ml. DC were enriched from the nonadherent population by den- amino acids, 1 ␮M sodium pyruvate, 2 mM L-glutamine, 50 ␮g/ml gen- sity centrifugation over medium containing 14.5% (w/v) metrizamide (Sig- tamicin, 100 U/ml penicillin, 0.5 ␮g/ml Fungizone, and 50 ␮M 2-ME (35). ma) (25). The cells at the metrizamide solution/medium interface were CD hybridoma serum-free medium (Invitrogen) was used to produce mAbs washed three times in the respective medium before use. The resulting DC used in vivo to avoid serum protein contamination of Abs. Murine rGM- population was analyzed by flow cytometry (FACScan; BD Biosciences, CSF (sp. act., Ն5.0 ϫ 106 U/mg) and murine rIL-4 (6.1 ϫ 108 U/mg) were San Jose, CA) for the expression of cell surface markers characteristic of purified from overexpressing cell lines generated in our laboratory; murine DC. Each preparation was Ն80% positive for the coexpression of MHC II, rIFN-␥ (50 ␮g/ml; BD PharMingen, San Diego, CA) was used to prepare CD11c, CD40, CD80, and CD86 (data not shown). The cells did not ex- a standard curve for ELISAs. Human rIL-2 (18.0 ϫ 106 U/mg) was ob- press CD3 or CD19. tained from Chiron (Emeryville, CA). Ag pulsing of DC Cell lines After purification, DCs were resuspended to 1.0 ϫ 106 cells/ml in either C6VL (H-2b) is a radiation-induced T cell thymoma of the C57BL/Ka CM or AIM-V medium containing lysate from either C6VL T cell lym- background (36). C6VL was used as our tumor model in these studies. phoma, MBL-2 (control) T cell lymphoma, B16/F10 melanoma, kidney C6VL cells have a mature T cell phenotype expressing TCR-␣␤, Thy 1.2, tissue, or normal primary lymphocytes. The cell lysates were prepared by The Journal of Immunology 5229

subjecting 20.0 ϫ 106 cells/ml in CM or AIM-V to three cycles of rapid ing only irradiated C6VL or MBL-2 were assayed to control for cytokine freezing in liquid nitrogen and thawing at 55¡C. The lysates were spun at secretion by stimulator cells. Sandwich ELISAs for IL-4 and IFN-␥ were 5000 rpm to remove particulate cellular debris. The DCs were pulsed with performed as described in Caltag’s protocol for cytokine ELISA with mod- the lysates from three tumor cells per DC for 18 h (25). DC derived in CM ifications. Briefly, rat anti-mouse IFN-␥ or rat anti-mouse IL-4 capture Abs were pulsed with lysates of cells cultured in CM, while DC derived in at 0.25 ␮g/ml in 50 mM carbonate (50 ␮l/well), pH 9.5, was used to coat AIM-V were pulsed with lysates of cells cultured in AIM-V. After pulsing, wells of a 96-well Nunc microtiter plate. After washing, 100 ␮l of undi- the DC were collected, washed several times in HBSS, and resuspended in luted supernatant from each VPLN restimulation were added to the plates HBSS to 5.0 ϫ 106 cells/ml for delivery to mice. and serially diluted 1:2 over eight wells with PBS with 1% BSA. Wells with known amounts of rIFN-␥ or rIL-4 were included on the plates to Immunizations generate standard curves. Biotinylated second-stage rat Abs against either ␥ Lysate-pulsed DC were injected s.c. in the right flank of the mice biweekly. IL-4 or IFN- were used to detect bound cytokine. Streptavidin-HRP was used to detect bound second-stage Ab. The peroxidase reaction was per- Mice vaccinated before tumor challenge or for in vitro restimulation stud- ␮ ies were immunized one to three times with 1.0 ϫ 106 (Fig. 1) or 5.0 ϫ 105 formed in a solution containing 50 mM sodium citrate (pH 4.0), 150 g/ml (Figs. 2Ð7) DC. Tumor-bearing mice were given 7.5 ϫ 105 lysate-pulsed ABTS, and 0.01% H2O2.OD405Ð450 was measured on a Vmax Microplate Reader (Molecular Devices, Menlo Park, CA). Data are represented as the DC every 10 days starting 1 day after receiving a lethal number of C6VL Ϯ tumor cells (Fig. 8). average of triplicate samples SD. Statistics between groups were measured using Student’s t test. Statistical significance was reached at Tumor challenge p Ͻ 0.05. C6VL and MBL-2 tumor cells used for tumor challenges were expanded in In vivo depletion of CD8ϩ T cells vitro and then frozen in aliquots in liquid nitrogen. With the exception of ϩ vaccinating tumor-bearing mice, mice were challenged with tumor 14 days CD8 T cells were depleted in vaccinated mice using anti-CD8.2 mAb ␮ following the last vaccination. Two days before tumor challenge, an aliquot 2.43. Mice were injected i.p. with 0.5 ml HBSS containing 250 gofthe of tumor cells was thawed and cultured in CM for 48 h. The cells were mAb on days 6, 5, and 4 before tumor challenge and with four weekly Downloaded from injections thereafter. Nondepleted mice received the isotype-matched con- collected by centrifugation, washed, and counted using a hemocytometer. ϩ The viability of the tumor cells before tumor challenge was generally trol mAb SFR8-B6 on the same schedule. The level of CD8 T cell de- Ͼ98% by trypan blue exclusion. The cells were diluted in HBSS such that pletion and the maintenance of depletion were assessed by flow cytometry a lethal number of tumor cells could be delivered i.p. in 500 ␮l. The 100% using a non-cross-blocking anti-CD8 mAb, 53-5.8. The analysis was per- lethal doses for C6VL and MBL-2 were 1000Ð5000 and 5000 cells, re- formed on PBL, splenocytes, and lymph node cells 1 day before tumor spectively. Groups of 10 mice were used for survival analysis. Survival of challenge and on splenocytes and lymph node cells 1 day before each of the tumor-challenged mice was monitored for 60 days following tumor chal- four weekly injections following tumor challenge. http://www.jimmunol.org/ lenge. Survival curves were constructed according to the Kaplan-Meier method. Statistical significance between vaccine groups was determined Adoptive transfer of VPLN cells using the log-rank test and was achieved when p Ͻ 0.05. Groups of 15 donor mice were vaccinated twice with C6VL lysate-pulsed C6VL staining with anti-C6VL Abs DC or the control vaccine, MBL-2 lysate-pulsed DC. The DC vaccination consisted of 2.5 ϫ 105 DC injected s.c. in the right flank and 1.25 ϫ 105 Flow cytometric analysis of C6VL cells stained with hyperimmune serum DC injected in each hind footpad. Nine days after the last immunization, was used to detect anti-C6VL Abs. Serum samples were obtained before donor mice were sacrificed, and VPLN (right inguinal and both popliteal) vaccination and 10 days following the third vaccination with 5.0 ϫ 105 were harvested. The lymphocytes were processed gently into single-cell unpulsed or lysate-pulsed DC or 35 ␮g TCR:KLH with QS-21. Serum suspensions and passed through 70-␮m pore size nylon mesh filters to samples from 10 mice from each experimental group were pooled, diluted remove cell clumps and debris. B cells were depleted using a nylon wool 1:10 in PBS to a volume of 50 ␮l, and incubated in polystyrene tubes with column. The B cell-depleted lymphocytes were washed twice with HBSS. by guest on October 3, 2021 5.0 ϫ 105 C6VL tumor cells for 30 min on ice. As a positive control, mAb Lymphocytes were analyzed pre- and post-nylon wool depletion to deter- ϩ ϩ 124-40 was added to preimmune serum at a concentration of 1.0 ␮g/1.0 ϫ mine B and T cell composition, as determined by CD3 and CD19 pop- 106 cells. The cells were washed with PBS plus 1% FBS and C6VL-bound ulations. Five million T cell-enriched VPLN in 200 ␮l HBSS were injected IgG and IgM were detected with 1.0 ␮g/1.0 ϫ 106 cells FITC-conjugated i.v. into groups of 10 naive recipients that were exposed to a sublethal dose goat anti-mouse IgGϩIgM (HϩL) Ab. The cells were washed three times of whole-body gamma irradiation (400 rad) 1 day previously. Immediately with PBS plus 1% FBS, fixed in 200 ␮l of 2% paraformaldehyde (w/v), and after injection, 3.0 ϫ 104 U of human rIL-2 in 200 ␮l HBSS were injected analyzed by flow cytometry using a FACScan (BD Biosciences). i.p. into each recipient and control mouse. IL-2 injections continued every 12 h for 4 days (eight total doses) (38). One day following the last IL-2 In vitro proliferation assay injection, mice were challenged with a lethal dose of C6VL tumor and followed for survival. Mice were vaccinated s.c. twice with 5.0 ϫ 105 C6VL lysate-pulsed DC, biweekly. Lymph nodes from untreated mice or vaccine-primed lymph nodes (VPLN) were harvested 10 days following the second vaccine, pro- Results cessed gently into single-cell suspensions, and passed through 70-␮m pore C6VL lysate-pulsed DC vaccines are more effective than TCR Id size nylon mesh filters to remove cell clumps or debris. Triplicate samples protein vaccines of 1.5 ϫ 105 cells in CM were seeded in 96-well, flat-bottom plates. The lymphocytes were stimulated with irradiated C6VL (2500 rad) or MBL-2 Prior experiments have shown that C6VL TCR Id protein vaccines (7500 rad) at stimulator:responder (S:R) ratios of 1:2, 1:10, and 1:50. Con are able to stimulate a protective immune response. To determine A stimulation was used as a positive proliferation control and was titrated whether lysate-pulsed DC vaccines were also effective in stimu- ␮ from 0.25 to 0.01 g/ml in triplicate wells. Lymphocytes from untreated lating anti-tumor immunity against C6VL, groups of 10 mice were mice were used as a negative control. The cells were incubated at 37¡Cin ϫ 6 a humidified incubator for 4 days, with 1 ␮Ci/well [3H]thymidine added for vaccinated three times with either 1.0 10 C6VL lysate-pulsed the last 16 h. Cells were harvested onto glass-fiber filters using a FilterMate DC (C6VL-DC) or TCR Id:KLHϩQS-21. Unpulsed DC and cell harvester (Packard Bioscience, Meriden, CT) and counted on a scin- HBSS vaccines were administered to groups of mice as controls. tillation counter (Packard Bioscience). The stimulation index (SI) for each Fourteen days following the third vaccine, mice received a lethal sample was calculated as: [([3H]thymidine incorporation (cpm) with stimulation Ϫ cpm without stimulation)/cpm without stimulation] and is rep- dose of C6VL and were monitored for survival. C6VL-DC vac- resented as the mean of triplicate samples Ϯ SD. Statistics between groups cines stimulated high levels of anti-tumor immunity in vaccinated at each S:R ratio were measured at the 95% confidence level using Stu- mice, resulting in 100% survival of the mice (Fig. 1). The protec- dent’s t test. tion generated by C6VL-DC vaccines was superior to the response In vitro cytokine secretion assay generated by TCR Id protein vaccines ( p ϭ 0.0042). TCR Id: KLHϩQS-21 vaccination did, however, still significantly enhance VPLN cells were collected as described above. VPLN (2.0 ϫ 106)in1ml CM were plated in triplicate wells of a 24-well plate with either 1.0 ϫ the survival of tumor-challenged mice compared with HBSS con- 106/well irradiated C6VL or irradiated MBL-2, 0.25 ␮g/ml Con A, or no trols ( p Ͻ 0.0001) as was previously reported (11). The survival of stimulation (medium only) and incubated at 37¡C for 96 h. Wells contain- C6VL-DC recipients was significantly increased compared with 5230 DC-BASED IMMUNOTHERAPY OF T CELL LYMPHOMA

FIGURE 1. C6VL-DC vaccines stimulate a superior protective immune response compared with TCR Id vaccines. Groups of 10 mice were vac- Downloaded from cinated three times with 1.0 ϫ 106 C6VL-DC, unpulsed DC, TCR Id: KLHϩQS-21, or HBSS. Two weeks following the third vaccination, mice were injected with 5000 C6VL cells and were monitored for survival.

recipients of unpulsed DC ( pϽ0.0001), which did not enhance the http://www.jimmunol.org/ survival of mice compared with HBSS injections ( p ϭ 0.214).

Protection from tumor challenge is lysate specific To determine whether the anti-C6VL tumor protection conferred by DC vaccines was lysate specific, groups of 10 mice were vaccinated three times with 5.0 ϫ 105 C6VL-DC or control lysate- pulsed DC. Control lysates were prepared from an irrelevant T cell lymphoma (MBL-2), an irrelevant tumor (B16/F10 melanoma), by guest on October 3, 2021 normal lymphocytes, or irrelevant tissue (kidney). Unpulsed DC vaccines and HBSS injections were included as negative controls (Fig. 2A). Mice were challenged with a lethal dose of C6VL 14 days following the third vaccine. C6VL-DC stimulated high levels of protection compared with unpulsed DC ( p ϭ 0.0002) or HBSS injection ( p ϭ 0.0018). DC pulsed with control tumor or tissue lysates did not stimulate a protective immune response against FIGURE 2. C6VL-DC vaccines stimulate both lysate- and tumor-spe- C6VL, and survival was indistinguishable among the groups cific survival of mice. A, Groups of 10 mice were vaccinated three times ( p Ͼ 0.6). with 5.0 ϫ 105 DC pulsed with lysates prepared from C6VL, irrelevant The lysate specificity of the tumor protection was further eval- lymphoma cells, normal lymphocytes, melanoma cells, or kidney tissue. uated in a cross-challenge experiment. Groups of 20 mice were Unpulsed DC vaccines and HBSS were also administered. Two weeks vaccinated twice with 5.0 ϫ 105 C6VL-DC or MBL-2-DC. Two following the third vaccine, mice were challenged with 5000 C6VL cells weeks following the last vaccine, 10 of the mice from each vaccine and were monitored for survival. B, Groups of nine mice received two ϫ 5 group were injected with a lethal dose of C6VL, while the other 10 vaccines of 5.0 10 DC pulsed with either C6VL or MBL-2 lysate. Two weeks following the last vaccine, mice were challenged with a lethal num- were injected with a lethal dose of MBL-2. C6VL-DC protected ber of either C6VL (1000 cells) or MBL-2 (5000 cells) and were monitored eight of 10 mice challenged with C6VL. When C6VL-DC vaccine for survival. recipients were challenged with MBL-2, the survival of the mice was significantly reduced compared with that of C6VL-challenged mice ( p ϭ 0.014; Fig. 2B). Similarly, MBL-2-DC-vaccinated mice were poorly protected against C6VL as opposed to MBL-2 chal- C6VL-DC and still protect the immunized mice (Fig. 3). Groups of lenge ( pϽ0.001; Fig. 2B). The lack of C6VL protection by MBL- 10 mice were vaccinated with 5.0 ϫ 105 C6VL-DC one, two, or 2-DC vaccines was not due to a lack of an immune response stim- three times before tumor challenge with C6VL. Mice immunized ulated by the vaccine, as MBL-2-DC vaccination protected 100% with three vaccines of 5.0 ϫ 105 MBL-2-DC were included as a of mice that were lethally challenged with MBL-2 (Fig. 2B). control group. Two and three vaccines of 5.0 ϫ 105 C6VL-DC stimulated equal levels of protection against lethal tumor challenge Two and three C6VL lysate-pulsed DC vaccines stimulate equal ( p ϭ 0.54), which was significantly different from the control vac- levels of protection cine ( p ϭ 0.0002). C6VL-DC given once before tumor challenge Three immunizations with 5.0 ϫ 105 C6VL-DC resulted in nearly stimulated intermediate levels of survival (40%) that were signif- all the mice surviving a lethal inoculation of tumor cells (Fig. 2A). icantly lower than those observed in the two- or three-vaccine We sought to determine whether we could use fewer injections of groups ( p Ͻ 0.05). The Journal of Immunology 5231

105 C6VL-DC, MBL-2-DC, or unpulsed DC (Fig. 4A). Mice were also vaccinated with TCR Id:KLHϩQS-21 as a positive control for C6VL-specific Ab production (Fig. 4B). Preimmune sera were obtained before the first vaccination, and hyperimmune sera from 10 mice/group were collected and pooled 10 days following the third vaccine. Preimmune serum spiked with anti-C6VL TCR V␣ mAb 124-40 was used as a positive control for C6VL staining. Three injections of TCR Id:KLHϩQS-21 stimulated a high titer of anti- C6VL Abs. Hyperimmune serum from mice vaccinated with C6VL-DC had only a small amount of Abs that bound to C6VL compared with preimmune serum staining (mean log fluorescence (MLF), 6.69 compared with 3.48). The same modest increase in C6VL staining was observed with the hyperimmune serum from mice vaccinated with unpulsed DC (MLF 11.11) and MBL-2-DC (MLF 5.52). An ELISA using plate-bound protein from C6VL lysate also showed low amounts of nonspecific Ab binding to C6VL proteins (data not shown).

FIGURE 3. Two and three C6VL lysate-pulsed dendritic cell vaccines C6VL lysate-pulsed DC stimulate tumor-specific cellular Downloaded from provide equal levels of tumor protection. Groups of 10 mice were vacci- responses nated with 5.0 ϫ 105 C6VL-DC (one, two, or three vaccines) or a control vaccine (three vaccines). Two weeks following the last vaccination, the mice To characterize the cellular immune response generated by were challenged with 5000 C6VL cells and were monitored for survival. C6VL-DC vaccines, VPLN were harvested from mice and a standard cell proliferation assay was performed (Fig. 5A). VPLN from mice vaccinated twice with 5.0 ϫ 105 C6VL-DC were stimulated http://www.jimmunol.org/ C6VL lysate-pulsed DC do not stimulate significant humoral in vitro with either irradiated C6VL or MBL-2 control T cell lym- responses against the tumor phoma cells. Background proliferation values for each stimulation group were determined for triplicate wells containing responder ϩ As reported previously, TCR Id:KLH QS-21 vaccines stimulate cells only. Naive lymphocytes were stimulated with irradiated high titers of anti-C6VL TCR Id-specific Abs (10, 11). To assess C6VL as a negative proliferation control. Con A stimulation of whether C6VL-DC vaccines stimulated C6VL-specific Ab produc- VPLN cells was included as a positive proliferation control. Stim- tion, hyperimmune serum from vaccinated mice was used to stain ulation of VPLN cells with irradiated C6VL tumor cells resulted in ϫ C6VL cells (Fig. 4). Mice were vaccinated three times with 5.0 high levels of proliferation at each S:R ratio. The SI were 12.5 Ϯ 1.7, 8.75 Ϯ 0.61, and 5.53 Ϯ 0.87 for ratios of 1:2, 1:10, and 1:50, by guest on October 3, 2021 respectively. Stimulation of vaccine-primed cells with MBL-2 resulted in moderate levels of proliferation (SI of 4.27 Ϯ 0.26, 3.69 Ϯ 0.73, and 2.16 Ϯ 0.19 for ratios of 1:2, 1:10, and 1:50, respectively) that were not statistically different from the negative control ( p ϭ 0.67, 0.08, and 0.07 for ratios of 1:2, 1:10, and 1:50, respectively). Stimulation of VPLN cells with irradiated C6VL resulted in significantly higher levels of proliferation than were observed upon stimulation with MBL-2 ( p ϭ 0.0041, Ͻ0.0001, and 0.020 for ratios of 1:2, 1:10, and 1:50, respectively). No proliferation of irradiated tumor cells was detected. Cytokine ELISAs were used to measure cytokine secretion by VPLN stimulated by C6VL. Triplicate samples of VPLN cells from mice vaccinated twice with 5.0 ϫ 105 C6VL-DC were stimulated in vitro with irradiated C6VL or MBL-2 cells at a ratio of two VPLN cells to one irradiated tumor cell. Con A stimulation was included as a positive control for IFN-␥ secretion. Unstimu- lated VPLN cells served as a negative control. Irradiated tumor cells were incubated without responder cells to control for stimulator cell cytokine secretion. Stimulation of VPLN with irradiated C6VL resulted in high levels of IFN-␥ secretion (66.7 Ϯ 5.4 ng/ ml) compared with stimulation with MBL-2 (21.4 Ϯ 3.4 ng/ml; p ϭ 0.022). IL-4 was also specifically secreted by VPLN upon ex FIGURE 4. DC vaccines stimulate low levels of anti-C6VL Abs. Pre- vivo stimulation with irradiated C6VL (Ͻ1 ng/ml), although at immune (shaded curve) and hyperimmune serum samples from mice vac- ϳ ␥ ϫ 5 levels 80-fold lower than IFN- (data not shown). Stimulation of cinated three times with 5.0 10 unpulsed (solid light gray line), C6VL ␥ lysate-pulsed DC (solid black line), or MBL-2 lysate-pulsed DC (solid gray VPLN cells with irradiated MBL-2 cells resulted in IFN- secre- line) (A) or TCR Id:KLH (35 ␮g) with 10 ␮g QS-21 (solid black line) (B) tion not statistically different from that observed in the unstimu- were used to stain cultured C6VL cells. As a positive control for both lated VPLN cultures ( p ϭ 0.11) and did not result in secretion of panels, an mAb specific for the V␣-chain of the C6VL TCR was added to IL-4 above the limit of detection of the ELISA (0.11 ng/ml; data preimmune serum (dotted gray line). C6VL-bound IgG and IgM were detected not shown). Irradiated tumor cells incubated without responder using FITC-conjugated goat anti-mouse IgG and IgM secondary mAb. cells did not secrete measurable levels of IFN-␥ or IL-4. 5232 DC-BASED IMMUNOTHERAPY OF T CELL LYMPHOMA

FIGURE 6. Transfer of T cell-enriched VPLN to naive mice confers protection. Groups of 10 mice (sublethally irradiated) received 5.0 ϫ 106 Downloaded from T cell-enriched lymph node cells from mice vaccinated with C6VL-DC or MBL-2-DC followed by 4 days of IL-2. On day 5 the mice received a lethal number of C6VL cells and were monitored for survival. Groups of 10 mice vaccinated twice with C6VL-DC or HBSS were used as positive and negative controls, respectively. http://www.jimmunol.org/ could be solely conferred by immune T lymphocytes. VPLN were harvested from donor mice vaccinated twice with 5.0 ϫ 105 C6VL-DC. This cell population contained 38% CD19ϩ B cells and 49% CD3ϩ T cells as determined by ﬂow cytometry (data not shown). After depleting B cells with a nylon wool column, the cells were Ͼ95% T cells (data not shown). Five million T cell- enriched VPLN were transferred i.v. into sublethally irradiated naive recipient mice. Negative control mice were injected with 5.0 ϫ by guest on October 3, 2021 106 of similarly treated vaccine-primed lymphocytes from MBL- 2-DC-vaccinated donor mice. Groups of 10 mice that were injected with C6VL-DC or HBSS but were not transferred served as positive and negative vaccine controls, respectively. All recipient and control mice received 30,000 U of recombinant human IL-2 be- ginning immediately after the transfer continuing every 12 h for eight total injections, based on reports that IL-2 induces the proliferation of the transferred cells and prolongs their survival in vivo (38). Four days after transfer, lymphocytes were isolated from one FIGURE 5. Vaccination with C6VL-DC stimulates tumor-speciﬁc pro- mouse from each group and used in a proliferation assay. The liferation and cytokine release by VPLN cells. Mice were vaccinated twice proliferative responses to irradiated C6VL or MBL-2 stimulation with 5.0 ϫ 105 C6VL-DC. Lymph nodes were harvested 10 days following observed in transfer recipients were less than one-half the re- the second vaccine and analyzed for their proliferative response (A) and cytokine secretion (B). A, VPLN were stimulated with irradiated C6VL or sponses in representative mice vaccinated directly with C6VL-DC MBL-2 (control lymphoma) cells at S:R ratios of 1:2, 1:10, and 1:50. Con or MBL-2-DC (data not shown). Five days following transfer or 1 A (positive control) was titrated from 0.25Ð0.01 ␮g/ml. The SI for each day after the IL-2 injections were completed, mice were chal- sample was calculated as: [(cpm with stimulation Ϫ cpm without stimu- lenged with a lethal dose of C6VL tumor (Fig. 6). Recipient mice lation)/cpm without stimulation] and is represented as the mean of tripli- that received immune lymphocytes from C6VL-DC were protected cate samples Ϯ SD. B, VPLN were stimulated with irradiated C6VL, ir- from C6VL challenge at a level near that in the positive control radiated MBL-2, Con A (positive control), or medium only (negative mice ( p ϭ 0.22), demonstrating that the protective immunity could control). After 96 h, culture media were assayed by sandwich ELISA for be transferred with 5.0 ϫ 106 vaccine-primed lymphocytes. Re- ␥ IL-4 (data not shown) and IFN- secretion. Bars represent the mean of cipient mice that received immune lymphocytes from MBL-2-DC triplicate samples Ϯ SD. Cytokine secretion by irradiated tumor cells was were not protected from C6VL challenge compared with recipients below the limits of detection in all cases. of C6VL-DC-primed lymphocytes ( p ϭ 0.00043), as their survival was equivalent to that of HBSS-injected control mice ( p ϭ 0.71). The role of CD8ϩ effector cells in mediating protective anti- T cells are essential for tumor protection tumor immunity was assessed by depleting CD8ϩ T cells in The importance of the T cell-mediated immune response in pro- C6VL-DC-immunized mice with an anti-CD8 mAb (Fig. 7). Mice tection against C6VL tumor in vivo was determined by two dif- were immunized twice with 5.0 ϫ 105 lysate-pulsed DC and then ferent approaches. An adoptive transfer experiment was performed treated with either an anti-CD8 mAb or an isotype-matched control (Fig. 6) to determine whether tumor protection against C6VL mAb. CD8ϩ cells were depleted (Ͼ98%) in peripheral blood, The Journal of Immunology 5233

FIGURE 7. CD8ϩ T cells are required for tumor protection. Groups of FIGURE 8. Vaccination with C6VL lysate-pulsed DC enhances the sur- Downloaded from 10 mice were vaccinated twice with 5.0 ϫ 105 C6VL-DC and treated with vival of tumor-bearing mice. Naive mice were challenged with a lethal an isotype-matched irrelevant mAb (SFR8-B6; F), C6VL-DC and treated dose of C6VL. Twenty-four hours following tumor challenge, mice re- with a CD8 T cell-depleting mAb (2.43; E), or MBL-2-DC and treated ceived their ﬁrst vaccine of TCR Id:KLH (35 ␮g/mouse) with QS-21 (10 with an isotype-matched irrelevant mAb (SFR8-B6; X). Two weeks fol- ␮g/mouse), C6VL lysate-pulsed DC, or control lysate-pulsed DC (7.5 ϫ 5 lowing the last vaccination, mice were challenged with 5000 C6VL cells 10 /mouse). The vaccines continued every 10 days for three vaccines. The and were monitored for survival. CD8ϩ T cells were depleted before tumor mice were monitored for survival for 60 days following tumor challenge.

challenge, and depletion was maintained for 30 days following tumor http://www.jimmunol.org/ challenge.

protection (11). Although vaccination with TCR Id:KLH in QS-21 enhanced the survival of mice against a subsequent tumor chal- spleen, and lymph nodes, and the depletion was maintained for 30 lenge, it was not effective therapeutically in tumor-bearing mice days after tumor challenge. The isotype-matched irrelevant mAb (Fig. 8). We hypothesized that a vaccine designed to stimulate ϩ had no effect on the CD8 T cell population of mice. Mice vac- potent cell-mediated responses against a broader array of tumor ϩ cinated with C6VL-DC that were not depleted of CD8 T cells Ags would be more efficacious. were highly protected from a C6VL challenge compared with mice DC are widely known to be potent inducers of CD4ϩ and CD8ϩ ϩ by guest on October 3, 2021 vaccinated with MBL-2-DC ( p ϭ 0.000003). Depletion of CD8 T cell-mediated responses against tumor cells (14). In addition, T cells completely abrogated tumor protection in mice vaccinated evidence suggests that vaccination with a mixture of Ags may with C6VL-DC ( p ϭ 0.000003). yield enhanced anti-tumor immunity as well as decrease the pos- sibility of tumor escaping the immune response (30, 32). The gen- C6VL lysate-pulsed DC vaccines are therapeutic in eration of a vaccine for T cell lymphoma consisting of multiple tumor-bearing mice known epitopes is currently limited due to the lack of well-char- To determine whether C6VL-DC vaccines were effective in treat- acterized, tumor-specific Ags. However, loading DC with tumor ing pre-existing C6VL tumors, groups of 10 unvaccinated mice RNA, DNA, or cell lysates allows a similar effect without neces- were challenged with a lethal dose of C6VL and immunized for the sitating the discovery of each Ag. In this study we directly com- first time 1 day thereafter. The mice were vaccinated with 7.5 ϫ pare the abilities of TCR Id:KLHϩQS-21 and tumor lysate-pulsed 105 C6VL-DC, MBL-2-DC, or TCR Id:KLH ϩ QS-21 every 10 DC vaccines to stimulate protective immunity against the murine days for three immunizations. C6VL-DC vaccines significantly en- T cell lymphoma, C6VL. We report that vaccination of mice with hanced the survival of tumor-bearing mice compared ϩ mice vac- C6VL lysate-pulsed DC stimulates potent anti-tumor protection cinated with MBL-2-DC ( p ϭ 0.05) or TCR Id:KLH ϩ QS-21 that surpasses immunity stimulated by TCR Id:KLHϩQS-21 vac- ( p ϭ 0.03), with 40% of the tumor-challenged mice surviving cines (Fig. 1). (Fig. 8). TCR Id protein vaccine recipients were not protected from Our goal in using C6VL lysate-pulsed DC to stimulate anti- existing C6VL tumor. tumor immunity was to have a broader array of tumor-specific Ags at which to target an immune response. However, a point that Discussion cannot be overlooked is that tumor lysates contain self-Ags as well Tumor-derived TCR Id protein is a tumor-specific Ag that has as tumor-specific Ags. The benefits of using whole tumor cell ly- been successfully exploited in the development of active immu- sates as Ag sources for loading DC have been demonstrated in notherapy protocols against defined epitopes of the murine T cell many cases without the development of autoimmune responses lymphoma, C6VL (10). Vaccination with TCR Id:KLHϩQS-21 toward self-Ags (25, 39). Recently, Vierboom et al. (40) demon- conferred significant levels of protection from an otherwise lethal strated that expression of some self Ags can serve as targets for tumor challenge (11). Mechanistically, TCR Id vaccines promoted CTL-mediated destruction without demonstrable damage to nor- both cellular and humoral immune responses when they were ad- mal tissues. Notwithstanding, skin depigmentation (41), autoim- ministered with the chemical adjuvant QS-21. Despite stimulating mune diabetes, and myocarditis (42) have been induced in mice high levels of TCR Id-specific Ab production, the presence of Abs vaccinated with tissue-specific Ags. C6VL is a CD4ϩ T cell ma- in hyperimmune serum did not correlate with tumor protection in lignancy. Therefore, we were particularly interested in whether vaccinated mice. CD8ϩ T cells were absolutely required for tumor lymphocyte populations were depleted in our immunized mice. 5234 DC-BASED IMMUNOTHERAPY OF T CELL LYMPHOMA

Based on our data, C6VL-DC vaccines did not promote the de- activated in the spleens or lymph nodes of C6VL-DC recipients, velopment of autoreactivity against self-CD4ϩ T cells or other and the survival of vaccinated mice was not influenced by the immune cell populations in the spleen, lymph nodes, or peripheral depletion of NK1.1ϩ cells before C6VL challenge (data not blood (data not shown). shown). The role of CD4ϩ T cells in the anti-tumor immunity Immunizations with C6VL-DC vaccines stimulated lysate-spe- generated by C6VL-DC vaccines was not tested in our experi- cific survival in vivo (Fig. 2). Injections of DC pulsed with mel- ments, as anti-CD4 Abs would also deplete C6VL cells used for anoma lysate, normal lymphocyte lysate, or kidney tissue lysate tumor challenge. did not protect mice from C6VL challenge, indicating that C6VL Immunotherapeutic approaches for T cell lymphoma are likely lysate contains antigenic epitopes capable of stimulating C6VL to be most effective for the clearance of minimal residual disease protection that are not highly expressed in nonmalignant tissues or in patients following conventional therapies. To more closely by all malignancies. Vaccination of mice with DC pulsed with model the clinical situation of a patient with minimal residual dis- lysates prepared from the control T cell lymphoma line, MBL-2, ease, we determined the efficacy of the C6VL-DC vaccine in mice did not protect mice against tumor challenge. Thus, the Ags stim- already carrying a lethal number of C6VL cells. Naive mice or ulating protective anti-tumor immunity against C6VL are not recipients of control vaccines succumb to lethal dose C6VL chal- shared by all T cell lymphoma tumors. Indeed, mice vaccinated lenge in ϳ30 days. Despite the aggressive nature of C6VL growth, with MBL-2 lysate-pulsed DC were protected against subsequent C6VL-DC vaccines were able to stimulate a protective immune MBL-2 tumor challenge, but not against C6VL challenge (Fig. response quickly enough to significantly enhance the survival of 3B), demonstrating that tumor lysate-pulsed DC vaccines could be tumor-bearing mice (Fig. 8). These data indicate that lysate-pulsed efficacious in stimulating lysate-specific, anti-tumor responses in DC vaccines may have clinically relevant therapeutic potential in other T cell lymphomas as well. Similar findings have been re- patients with pre-existing disease. Future experiments using this Downloaded from ported for the use of primary multiple myeloma (33) and B cell vaccine protocol will focus on the use of cytokine adjuvants to chronic lymphocytic leukemia (34) lysate-pulsed DC to stimulate enhance the anti-tumor activity of C6VL-DC vaccines in the set- patient CTL activity against autologous tumors in vitro. In these ting of pre-existing T cell lymphoma. studies primary tumor lysate-pulsed DC stimulated CTL activity Collectively, C6VL-DC vaccines have proven to stimulate im- against autologous malignant cells, but not against autologous non- mune responses superior in their potential to prevent future tumor malignant cells and rarely against allogeneic tumor cells. Collec- growth and clear existing C6VL T cell lymphoma compared with http://www.jimmunol.org/ tively, these data argue that lymphoma lysate-pulsed DC as ther- previously reported TCR Id protein and TCR Id adenoviral vac- apeutics would require the preparation of vaccines on a per-patient cines. The studies presented herein provide evidence that vaccina- basis. tion with T cell lymphoma lysate-pulsed DC may allow enhanced The finding that 100% of the mice vaccinated three times with efficacy in the clinical treatment of T cell malignancies and result 1.0 ϫ 106 lysate-pulsed DC survived subsequent tumor challenge in increased disease-free survival rates of patients without neces- was surprising (Fig. 1). Vaccinating mice with DC derived and sitating the identification of tumor-specific Ags or patient-specific loaded with Ag under serum-free conditions ensured that protec- Id protein expression. tion from tumor challenge was not due to an immune response by guest on October 3, 2021 generated against FBS proteins on C6VL cells cultured in serum- Acknowledgments containing media (Figs. 2A,3,4,and6Ð8). Decreasing the number We thank A. Chang for helpful suggestions regarding the in vitro prolif- ϫ 5 of C6VL-DC delivered to mice to 5.0 10 for three vaccines did eration assay and adoptive transfer experiments. not result in a significant decline in tumor protection (Fig. 2A). ϫ 5 Furthermore, two vaccines of 5.0 10 C6VL-DC stimulated lev- References els of tumor protection equal to three vaccines (Fig. 3), demon- 1. Timmerman, J. M., and R. Levy. 2000. The history of the development of vac- strating that we could be even less aggressive in our vaccination cines for the treatment of lymphoma. Clin. Lymphoma 1:129. protocol. Based on these data, we modified our vaccination pro- 2. Hart, D. N., and G. R. Hill. 1999. Dendritic cell immunotherapy for cancer: ϫ 5 application to low-grade lymphoma and multiple myeloma. Immunol. Cell. Biol. tocol to two vaccines of 5.0 10 DC for the experiments de- 77:451. picted in Figs. 2B and 5Ð7. The delivery of one C6VL-DC vaccine 3. Campbell, M. J., W. Carroll, S. Kon, K. Thielemans, J. B. Rothbard, S. Levy, and before tumor challenge generated levels of protection similar to R. Levy. 1987. Idiotype vaccination against murine B cell lymphoma: humoral ϩ and cellular responses elicited by tumor-derived immunoglobulin M and its mo- those observed when three TCR Id:KLH QS-21 vaccines were lecular subunits. J. Immunol. 139:2825. delivered. Whether this result is indicative of an enhanced ability 4. Campbell, M. J., L. Esserman, N. E. Byars, A. C. Allison, and R. Levy. 1990. of DC to prime immune responses compared with TCR Id or of a Idiotype vaccination against murine B cell lymphoma: humoral and cellular re- quirements for the full expression of antitumor immunity. J. Immunol. 145:1029. broader immune response generated against multiple or more po- 5. Dyke, R. J., H. McBride, A. J. George, T. J. Hamblin, and F. K. Stevenson. 1991. tent Ags is unclear, and further work is being performed to exam- Idiotypic vaccination against B-cell lymphoma leads to dormant tumour. Cell. ine this question. Immunol. 132:70. 6. George, A. J., S. G. Folkard, T. J. Hamblin, and F. K. Stevenson. 1988. Idiotypic C6VL-DC vaccines did not stimulate strong humoral immune vaccination as a treatment for a B cell lymphoma. J. Immunol. 141:2168. responses against C6VL (Fig. 4A). Further, Ab production did not 7. Syrengelas, A. D., and R. Levy. 1999. 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CORRECTION

Erin Gatza and Craig Y. Okada. Tumor Cell Lysate-Pulsed Dendritic Cells Are More Effective Than TCR Id Protein Vaccines for Active Immunotherapy of T Cell Lymphoma. The Journal of Immunology 2002;169:5227–5235.

The authors of this paper neglected to identify the source of a reagent used in the study. The source of the reagent is stated below.

“The C6VL TCR Id vaccine used in this study was prepared by conjugating recombinant TCR Id protein to KLH and mixing the conjugate with QS-21. QS-21 was provided by Antigenics, Inc. (Framingham, MA) under a material transfer agreement.”