Gene-modified Dendritic Cells CD4 T Cells during ...

CD40 Cross-Linking Bypasses the Absolute Requirement for CD4 T Cells during Immunization with Melanoma Antigen Gene-modified Dendritic Cells Antoni Ribas, Lisa H. Butterfield, Saral N. Amarnani, et al. Cancer Res 2001;61:8787-8793.

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[CANCER RESEARCH 61, 8787– 8793, December 15, 2001]

CD40 Cross-Linking Bypasses the Absolute Requirement for CD4 T Cells during Immunization with Melanoma Antigen Gene-modified Dendritic Cells1 Antoni Ribas, Lisa H. Butterfield, Saral N. Amarnani, Vivian B. Dissette, Donald Kim, Wilson S. Meng, Gustavo A. Miranda, He-Jing Wang, William H. McBride, John A. Glaspy, and James S. Economou2 Divisions of Surgical Oncology [A. R., L. H. B., S. N. A., V. B. D., D. K., W. S. M., G. A. M., J. S. E.] and Hematology-Oncology [A. R., J. A. G.], and Departments of Biomathematics [H-J. W.], Experimental Radiation Oncology [W. H. M.], and Microbiology, Immunology, and Molecular Genetics [J. S. E.], University of California at Los Angeles, Los Angeles, California 90095

ABSTRACT Genetic immunization of mice with dendritic cells (DCs) engineered to express a melanoma antigen generates antigen-specific, MHC-restricted, CD4-dependent protective immune responses. We wanted to determine the role of CD4 cells and CD40 ligation of MART-1 gene-modified DC in an animal model of immunotherapy for murine melanoma. CD4 knockout (CD4KO) or antibody-depleted mice were immunized with DC adenovirally transduced with the MART-1 gene (AdVMART1/DC) with or without CD40 cross-linking. Tumor protection was absent in CD4depleted mice, but protection was reestablished when the CD40 receptor was engaged using three different constructs. Transduction of DCs with vectors expressing the Th1 cytokines interleukin (IL)-2, IL-7, or IL-12 could not reproduce the CD40-mediated maturation signal in this model. CD8 T-cell depletion in CD4KO mice immunized with CD40-ligated DCs abrogated the protective response. Pooled analysis of CD40 cross-linking of AdVMART1/DC administered to wild-type C57BL/6 mice revealed an overall enhancement of antitumor immunity. However, this effect was inconsistent between replicate studies. In conclusion, maturation of AdVMART1-transduced DCs through the CD40 ligation pathway can promote a protective CD8 T-cell-mediated immunity that is independent of CD4 T-cell help.

INTRODUCTION Genetic immunization with tumor antigen-transduced DCs3 generates antigen-specific cellular responses (1–5). This method takes advantage of the natural properties of DCs as optimal immunological adjuvants (6) and the ability of endogenous expression of tumor antigen genes to continuously provide antigenic epitopes for both MHC class I- and II-restricted expression (7, 8). Using MART-1 as a tumor antigen, we have demonstrated that adenovirally transduced DCs generate robust antigen-specific protective responses in mice (1, 8 –10) and in preclinical human experimental systems (7, 11). Mice immunized with AdVMART1/DC develop antigen-specific, MHC class I-restricted CTLs with a type 1 immune phenotype (1, 8). This response is mediated by the immunizing DCs and not by cross-priming (9), and there is an absolute requirement for an intact CD4 helper T-cell subset (8). Several hypotheses have been proposed to explain the nature of CD4mediated “help.” It has long been recognized that CD4 cells can Received 7/5/01; accepted 10/18/01. 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 This work was supported in part by NIH/National Cancer Institute Grants RO1 CA77623, RO1 CA79976, T32 CA75956, and K12 CA76905 (all to J. S. E.); the Stacy and Evelyn Kesselman Research Fund; and the Monkarsh Fund. A. R. is a recipient of the American Society of Clinical Oncology Career Development Award and the Richard Barasch Seed Grant Award from Stop Cancer. 2 To whom requests for reprints should be addressed, at Division of Surgical Oncology, Room 54-140 CHS, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-1782. Phone: (310) 825-2644; Fax: (310) 825-7575; E-mail: jeconomou@ mednet.ucla.edu. 3 The abbreviations used are: DC, dendritic cell; APC, antigen-presenting cell; IL, interleukin; CD4KO, CD4 knock-out; ATCC, American Type Culture Collection; MOI, multiplicity of infection; GM-CSF, granulocyte/macrophage-colony stimulating factor; ELISPOT, enzyme-linked immunospot.

generate a type 1 cytokine milieu (IL-2, IL-7, IL-12, and IFN-␥) that facilitates the activation of cytotoxic CD8⫹ T cells. A direct, lymphokine-dependent CD4-CD8 T-cell communication as a mediator of CD4 T-cell help has been formally demonstrated (12). However, the critical step is now thought to arise from a sequential three-cell interaction centered on the APC, most likely a DC. In this model, DCs present MHC class II-restricted antigens to CD4 cells, which in turn activate and fully mature the DCs, leading to increased surface molecule expression of MHC, costimulatory and adhesion molecules, and IL-12 secretion. In different preclinical models of viral infection and foreign antigen immunization, the main mediator of DC activation by CD4 cells is CD40 receptor engagement (13–15). Other surface molecule interactions, such as the tumor necrosis factor-related activation induced cytokine (TRANCE)-TRANCE receptor interaction (16, 17), may have similar effects. According to this model, activated DCs are then optimally equipped to send stimulatory signals to CD8 cells, which recognize a cognate antigen presented by MHC class I molecules on the surface of the DCs. To characterize the nature of CD4 T-cell help after genetic immunization with gene-modified DCs expressing an endogenous tumor antigen, we have examined the role of CD40 cross-linking in mice immunized with AdVMART1/DC. Using three different CD40 crosslinking constructs, antigen-specific protective cytotoxic responses are generated in mice genetically deficient of CD4 cells and in mice depleted of CD4 T-cell compartment using antibodies, as assessed by a challenge with the naturally MART-1-expressing murine melanoma B16. A beneficial effect similar to CD40 cross-linking is not mediated by increased DC production of type 1 cytokines such as IL-2, IL-7, or IL-12, suggesting that CD40 receptor engagement has effects other than increased type 1 cytokine production by the DCs. Therefore, our results demonstrate for the first time the critical role of CD40 crosslinking in the protective immunity generated by genetic immunization with gene-modified DCs. MATERIALS AND METHODS Mice and Cell Lines. C57BL/6 and C57BL/6-Cd4tm1Mak mice (both H-2b) were purchased from The Jackson Laboratory (Bar Harbor, ME) and were bred and kept in the pathogen-defined Animal Facility of the Division of Experimental Radiation Oncology at the University of California Los Angeles. Female mice, 5– 8 weeks of age, were used for all studies. Mice were handled in accordance with the University of California Los Angeles animal care policy. B16, a murine melanoma, and EL4, a murine lymphoma cell line, were obtained from the ATCC (Rockville, MD) and maintained in vitro in DMEM (Life Technologies, Inc.) with 10% FCS (Gemini Products, Calabasas, CA) and 1% (vol/vol) penicillin, streptomycin, and fungizone (Gemini; complete media). EL4(MART-1) was developed by transfection of the parental line with a plasmid (pRcCMVMART-1) carrying the MART-1 cDNA and neo resistance gene as described previously (9). Stably transfected cells were maintained in vitro under constant G418 selection (0.5 mg/ml; Life Technologies, Inc.). Recombinant Adenoviruses. AdVMART1, AdVIL-2, and AdVIL-7 are E1-deleted replication-deficient adenoviral vectors based on human type 5 adenoviruses. The construction and characterization of these vectors has been

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BYPASSING CD4 T-CELL HELP

described previously (1, 11, 18, 19). The adenovirus murine-IL-12.1 (AdVIL12) vector, a generous gift from Dr. Frank Graham (McMaster University, Hamilton, Ontario, Canada), has also been described previously (20). This vector contains the p35 and p40 subunit cDNAs of murine IL-12 in the early regions 1 (E1) and 3 (E3), respectively, of adenovirus type 5. In all cases, the transgenes are driven by the human cytomegalovirus early promoter/enhancer. Preparation of DCs, Adenoviral Transduction, and CD40 Ligation. DCs were differentiated from murine bone marrow progenitor cells by in vitro culture in GM-CSF and IL-4 as described by Inaba et al. (21) with modifications (1). In vitro cultured DCs were transduced in 15-ml conical tubes (Costar, Acton, MA) in a final volume of 1 ml of RPMI with 2% FCS, to which the virus stock was added at a MOI of 100 viral plaque-forming units per each DC. Transduction was carried out for 2 h at 37°C, after which time the DCs were washed extensively and resuspended in 0.2 ml of PBS/animal for injection into mice. Cell counts were determined using a hemocytometer with viability assessed by trypan blue exclusion. In all cases, viability exceeded 95%. Transduction of murine DCs with the replication-defective adenoviral vector AdVMART1 (AdVMART1/DC) between MOI of 1–100 results in MART-1 expression that persists for at least 5 days (1). The level of IL-2, IL-7, and IL-12 cytokine production by DCs transduced with cytokine-expressing adenoviral vectors was determined by ELISA. Two constructs were used for ex vivo CD40 ligation, a recombinant trimeric murine CD40-ligand (a generous gift from Kathleen S. Picha, Immunex, Seattle, WA) and the antimouse CD40 activating antibody HM40-3 (purchased from PharMingen, San Diego, CA). Day 8 MART-1-transduced DCs were resuspended in complete medium with 0.1 ␮g/ml of trimeric CD40-ligand (Immunex) and 0.1 ␮g/ml IFN-␥ (Genzyme, Cambridge, MA) and returned to tissue culture for an additional 48 h. Activation with the anti-CD40 activating antibody HM40 –3 (PharMingen) was performed following the protocol described by Ridge et al. (13). Briefly, day 8 DCs were first transduced with AdVMART1, exposed to heat-inactivated murine serum for 5 min, treated with HM40 –3 for 1 h, and followed by overnight incubation with a secondary cross-linking antibody (goat antihamster IgG; Caltag). Flow Cytometry Analysis. The following monoclonal antibodies were used for flow cytometry analysis: Ly-2 (53-6.7) antimouse CD8a; GL1 antimouse CD86 (B7.2); Ly-38 (1B1); antimouse MHC class II I-Ab (clone AF6-120-1); and antimouse MHC class I H2-Kb (all from PharMingen, San Diego, CA). Clone 1G10 antimouse CD80 (B7.1; Southern Biotechnology Associates, Birmingham, AL). Animal Studies. Mice were immunized on days 1 and 8 with 1–5 ⫻ 105 DCs/mouse administered s.c. in the right flank and challenged on the left flank 14 days after the last immunization with B16 (1 ⫻ 105 cells/animal). Cells used for tumor challenge were obtained from single-cell suspensions of progressively growing tumors in syngeneic mice to avoid the confounding effects of the presentation of medium- and serum-derived epitopes (1). Cell suspensions were washed extensively and injected into mice in a final volume of 0.2 ml of PBS/animal. Each treatment group contained 4 to 5 mice for in vivo tumor challenge studies and one mouse to obtain splenocytes for in vitro studies (ELISPOT, 51Cr release assays). In Vivo Antibody Treatment. In vivo monoclonal antibody ablation of CD8 (clone 2.43; ATCC TIB 210) or CD4 (clone GK1.5; ATCC TIB 207) T-cell subsets was performed by i.p. injection on days ⫺5, ⫺3, and ⫺1, before the first immunization (in one study with CD8 depletion, antibody administration was started after the vaccinations and before tumor inoculation), and every 6 days thereafter (250 ␮g of purified antibody/mouse/injection). Antibody suspensions were purified from hybridoma supernatants by passage through protein G columns according to the manufacturer’s instructions (Pierce, Rockford, IL). Eluted immunoglobulins were dialyzed against PBS and stored at 4°C at 1 mg/ml suspensions. CD4⫹ and CD8⫹ T-cell depletion was monitored by flow cytometric analysis of splenocytes at the day of tumor challenge. For in vivo CD40 activation, 100 ␮g/mouse/injection of the CD40activating antibody FGK45 (a gift from Dr. Stephen P. Schoenberger, La Jolla Institute of Allergy and Immunology, San Diego, CA) were given i.v. in 0.2 ml of PBS 24 h after the DC immunization (14). Cytotoxicity Assays. For in vitro microcytotoxicity assays, splenocytes form one mouse from each treatment group were harvested 14 days after the last immunization, depleted of RBCs by hypotonic lysis, restimulated in vitro with irradiated EL4(MART-1) at a 25:1 ratio for 96 h in the presence of 10 units/ml of IL-2, and assayed in a standard 4-h chromium release test (8 –10).

For each different target, samples were tested against their own maximum and spontaneous release. Cytokine Profile by ELISPOT. RBC-depleted splenocytes, restimulated in vitro for 48 h at the same conditions described above for cytotoxicity assays, were added in duplicate 3-fold dilutions to 96-well mixed cellulose plates (Multiscreen filtration system; Millipore, Bedford, MA) precoated with antiIFN-␥ or anti-IL-4 antibody (PharMingen) as described previously (8 –10). After 24-h incubation at 37°C, plates were washed and incubated at 4°C with secondary biotinylated antibody. On the next day, spot-forming colonies were developed by the addition of horseradish peroxidase avidin D (Vector Laboratories, Burlingame, CA), followed by color reaction using 3-amino-9-ethylcarbazole (Sigma Chemical Co., St. Louis, MO). Spots were counted under a dissecting microscope. Statistical Analysis. Differences in tumor development were assessed using the ␹2 or the Fisher exact test. Results of in vivo studies are presented as the mean and SE of tumor volumes in each treatment group. Mice completely protected from a tumor challenge are presented separate from mice that did develop tumors to allow correct assessment of the rate of tumor growth (1). If a treatment group is divided into mice with and without tumors in a tumor volume plot, the number of mice with (or without) tumor from that group over the total number of mice in the group is shown in parentheses. Significance is calculated using the t test (or the rank sum test in case of failing the Kolmogorov-Smirnov test for Normality). Graphs of tumor development over time are presented using Kaplan-Meier plots, with the significance calculated using the log-rank test or a pair-wise Cox model.

RESULTS Effects of CD40 Ligation on Surface Molecule Expression on the DCs. DCs harvested after 8 days of in vitro culture in GM-CSF and IL-4 were transduced at an MOI of 1:100 with the replicationdefective vector AdVMART1. After transduction, nonabsorbed adenoviral vector was washed, and cells were replated in complete medium with either trimeric CD40-ligand or CD40 activating antibody. Forty-eight h later, cells were harvested and evaluated by flow cytometry for several phenotypic markers (Table 1). Treatment of DCs with the CD40 activating antibody HM40 –3 induced a marked increase in surface expression of MHC class I and II molecules but had a less striking effect on the expression of the costimulatory molecules CD80 and CD86. Treatment of DCs with the trimeric murine CD40-ligand led to an increase in MHC class I, class II, CD80, and CD86 expression. CD40 Ligation Leads to Protection in CD4KO Mice. Immunization of mice with AdVMART1-transduced DCs leads to protective immunity to a MART-1-positive tumor challenge, with an absolute requirement for CD4 cells (8). This response is mediated by a preferential type 1 immunological response with IFN-␥ production and leads to the generation of antigen-specific CTLs. We investigated whether CD40 ligation would bypass the requirement for CD4 cell help. Fig. 1 shows the composite analysis of tumor development in seven independent studies using a total of 64 wild-type and 82 CD4KO mice. Mice were immunized twice at weekly intervals with AdVMART1/DC with or without CD40 ligation and were challenged 2 weeks later at the opposite flank with a single-cell suspension of murine MART-1-positive B16 cells. Unimmunized wild-type and CD4KO mice were used as control groups for tumor development. All

Table 1 Flow cytometry analysis of surface markers in CD40-cross-linked DCs

Kb I-Ab CD80 CD86 a

AdVMART1/DC

AdVMART1/DC ⫹ CD40 antibody

AdVMART1/DC ⫹ CD40-ligand

1304a 271 249 7854

4706 607 232 8486

2602 710 571 9216

Mean fluorescence.

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Fig. 1. Antitumor protection can be reestablished in CD4KO mice with CD40 crosslinking. Wild-type (F, Œ) and CD4KO (E, ‚, 䡺) mice were immunized twice s.c. with 5 ⫻ 105 AdVMART1/DC with or without CD40 cross-linking (0.1 ␮g/ml of CD40-ligand and 0.1 ␮g/ml IFN-␥ for 48 h) and challenged 14 days later with 1 ⫻ 105 B16 cells. Mice were followed for tumor development. Log-rank test analysis of tumor development curves demonstrates statistically significant differences between wild-type control and AdVMART1/DC immunized mice (P ⫽ 0.0001) and CD4KO unimmunized control mice or CD4KO mice immunized with AdVMART1/DC compared with AdVMART1/DCCD40L (P ⫽ 0.0001) in both comparisons. There is no significant difference between tumor development in wild-type mice immunized with AdVMART1/DC compared with CD4KO mice immunized with CD40-cross-linked AdVMART1/DC (P ⫽ 0.18).

mice in these control groups developed tumors that grew progressively. Wild-type mice immunized with AdVMART1/DC showed intermediate antitumor protection, with 53% of the mice developing tumors. The majority of CD4KO mice immunized with AdVMART1/DC developed s.c. B16 tumors at a rate indistinguishable to the unimmunized mice, confirming the requirement for CD4 cells in this animal model. At the end of the study period, 65% of the CD4KO mice immunized with CD40-ligated AdVMART1/DC were completely protected from a B16 tumor challenge. These data confirm that CD40 ligation in gene-modified DCs bypasses the requirement of CD4 cells in this murine antitumor protection model using an endogenously expressed antigen. Generation of an Antigen-specific Type 1 Cytotoxic Response in CD4KO Mice Immunized with CD40-ligated DCs. CD4KO mice were immunized twice with DCs transduced with AdVMART1 or a control adenovirus (AdVLacZ) with CD40 cross-linking. Two weeks later, groups of mice were challenged s.c. with B16 and tumor growth were followed, or their spleens were harvested and restimulated in vitro for ELISPOT and microcytotoxicity assays. The in vivo tumor growth curves in CD4KO mice revealed that only mice receiving CD40-ligated MART-1-expressing DCs benefited from immunization (Fig. 2A). In two replicate studies, vaccination with CD40-ligated DCs transduced with a similar adenoviral vector expressing LacZ as a control antigen did not lead to antitumor protection. Splenocytes harvested from immunized and unimmunized mice were restimulated in vitro with a MART-1-expressing syngeneic cell line EL4(MART1). After 48 h of restimulation, cells were assayed for the presence of IFN-␥-producing cells by an ELISPOT assay. CD4KO mice immunized with AdVMART1/DC did not have IFN-␥-producing cells after re-exposure to MART-1. CD4KO mice immunized with the irrelevant antigen LacZ-expressing DCs with CD40 cross-linking had background levels of IFN-␥-producing splenocytes. In seven replicate studies, CD4KO mice immunized with CD40-ligated MART-1expressing DCs had detectable IFN-␥-producing cells, but the number of these type 1 antigen-specific cells was always lower than in wild-type mice immunized with AdVMART1/DC (a representative

study is shown in Fig. 2B). MART-1 in vitro-restimulated splenocytes from AdVMART1/DC-vaccinated wild-type mice contained CTLs that lysed chromated B16 in microcytotoxicity assays (Fig. 2C). In five replicate studies, CD4KO mice immunized with AdVMART1/DC contained background amounts of CTLs, whereas splenocytes from mice immunized with CD40-ligated AdVMART1/DC lysed B16 cells at a level close to the wild-type mice (Fig. 2C). In summary, immunization of CD4KO mice with AdVMART1/DC with CD40 cross-linking leads to the generation of CTLs in an antigen-specific type 1 environment. Comparison of Different Methods of CD40 Activation to Bypass CD4 Cell Requirement. DCs treated in vitro with the antimouse CD40-activating antibody HM40-3 or mice receiving i.v. injection of another CD40-activating antibody (FGK45) have been shown to bypass the CD4 T-cell requirement for immunization in several animal models (13–15, 22, 23). We compared the activity of the recombinant trimeric mouse CD40-ligand with these two activating antibodies. Fig. 3 shows that similar levels of antitumor protection can be achieved with all three CD40 cross-linking strategies in CD4KO mice immunized with AdVMART1/DC. CD40 Cross-Linking Restores Protection after Antibodymediated CD4 T-Cell Depletion. To confirm that CD40 cross-linking in gene-modified DC substitutes for the presence of CD4 T cells, and that this was not an artifact found only in CD4 genetically depleted mice. Wild-type mice were depleted of CD4 T cells using an anti-CD4 antibody. Successful depletion of ⬎99% of CD4 cells was confirmed by flow cytometry (data not shown). When these mice were immunized with AdVMART1/DC, tumors grew at a rate indistinguishable from the unimmunized mice (Fig. 4). As is the case with CD4KO mice, CD4-depleted mice that received AdVMART1/DC treated with the anti-CD40 activating antibody exhibited a level of antitumor protection similar to mice with intact CD4 T-cell compartment immunized with AdVMART1/DC. Effects of CD40 Cross-Linking Could Not Be Replaced by IL-2, IL-7, or IL-12. To determine whether certain Th1 type cytokines could replace the beneficial effects of CD40 cross-linking (12), we compared the antitumor protection generated by CD40-cross-linked AdVMART1/DC to AdVMART1/DC cotransduced with IL-2, IL-7, or IL-12-expressing adenoviral vectors. Cytokine production by DCs transduced with AdVIL-2, AdVIL-7, and AdVIL-12 was 20 ␮g/106 DC/24 h, 2.6 ng/106 DC/24 h, and 10.3 ng/106 DC/24 h, respectively. In two replicate studies, tumor growth in CD4KO mice immunized with AdVMART1/DC coexpressing IL-2, IL-7, or IL-12 was indistinguishable from control mice (Fig. 5A). In these mice, there were background levels of IFN-␥-producing splenocytes when restimulated with MART-1 in vitro (Fig. 5B). Conversely, CD40 ligation in these same experiments led to substantial antitumor protection and splenocyte IFN-␥ production. Role of CD8 T Cells in CD4KO Mice Immunized with CD40Cross-Linked, Gene-modified DCs. CD4KO mice immunized with AdVMART1/DC with CD40 cross-linking were further depleted of CD8 T cells to determine the role of this T-cell subset in this model. In two separate studies, mice received i.p. injections of a purified anti-CD8-depleting antibody. In one of the studies, CD8 depletion was started before the first DC vaccination (induction phase; data not shown), whereas in the second study, CD8 depletion was done after DC immunizations but before tumor challenge (effector phase; Fig. 6). Protection to B16 tumor challenge was abrogated when CD4KO mice were depleted of CD8 T cells before or after the first immunization, suggesting a role of these cells in both the induction and effector phase of the immunization with CD40-cross-linked DCs.

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Fig. 2. AdVMART1/DC-induced immunity in CD4KO mice is antigen specific and mediated by a cytotoxic type 1 response. Wildtype (F, Œ) and CD4KO (E, ‚, 䡺, ƒ) mice were immunized twice s.c. with 5 ⫻ 105 AdVMART1/DC with or without CD40 cross-linking (0.1 ␮g/ml of CD40-ligand and IFN-␥ for 48 h). A, tumor development and growth in mice challenged 14 days later with 1 ⫻ 105 B16 cells. As described in “Materials and Methods,” mice completely protected from B16 tumor challenge are presented separately from the rest of the group, and each divided line from a single group has the number of mice it represents in parentheses. Wild-type control versus AdVMART1/DC, P ⫽ 0.01; CD4KO AdVMART1/DC-CD40L versus control P ⫽ 0.005, versus AdVLacZ/DC-CD40L, P ⫽ 0.03, versus AdVMART1/DC, P ⫽ 0.03. B, IFN-␥-producing cells in splenocytes restimulated for 48 h with EL4(MART1) assayed in an ELISPOT assay. C, chromated B16 target cell lysis by MART-1-restimulated splenocytes. Wild-type AdVMART1/DC versus control, P ⫽ 0.0002; CD4KO AdVMART1/DC-CD40L versus control, P ⬍ 0.0001, versus AdVLacZ/DC-CD40L, P ⫽ 0.0002, versus AdVMART1/DC, P ⫽ 0.03. Bars, S.E.M.

Inconsistent Augmentation of Antitumor Protection in WildType Mice Immunized with CD40-Cross-Linked DCs. We performed several studies to test whether treating the DCs with CD40 cross-linking would increase the ability to generate antigen-specific responses in wild-type mice. Fig. 7 shows an actuarial plot of tumor development with pooled data from eight studies where AdVMART1/DC with or without CD40 antibody-mediated activation were administered to immunocompetent C57BL/6 mice. Overall, immunization with CD40 cross-linked AdVMART1/DC has a trend toward lower rate of tumor development compared with immunization with AdVMART1/DC alone. Comparison of tumor development curves reveal a significant improvement in tumor development in pair-wise comparisons using a Cox model (risk ratio, 1.763; 95% confidence interval, 1.0005–3.090; P ⫽ 0.048). However, the greater in vivo protection to a B16 tumor challenge is heavily dependent on four studies with significant CD40-enhanced protection, whereas in the other four replicate studies there was no effect related to CD40 cross-linking. Similar inconsistent results were noted in IFN-␥ ELIS-

POT, cytotoxicity assays, and tumor protection studies using trimeric CD40-L (data not shown). In these studies, the degree of DC maturation by phenotypic analysis did not correlate with the ability to enhance protection in wild-type mice (data not shown). DISCUSSION In many murine models of anticancer immunotherapy, CD4 T cells are essential in the induction and maintenance of cytotoxic responses mediated by CD8 T cells (24 –29). Three possible pathways of CD4mediated help have been described: CD40-dependent DC activation (13–15, 22, 23), CD40-independent DC maturation through other tumor necrosis factor superfamily receptors (TRANK/RAF; Refs. 16, 17), and direct lymphokine-dependent CD4-CD8 cell communication (12). Additionally, certain viral infections and cross-priming models induce CD8-mediated cytotoxic responses in the absence of CD4 T cells (30, 31), and in some instances CD4-mediated protective antitumor responses can be generated in the absence of CD8 cells (32).

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These different cell subpopulation requirements for the generation of antigen-specific cellular responses seem to be dependent on the model, the characteristics of the antigen, and its level of surface expression on APCs, as well as the immunization method. The mechanism of CD4-mediated help when immunizing with genetically modified DCs had not been reported before this study. When immunizing with DCs genetically modified to express two different tumor antigens (MART-1 and AFP), we have reported previously that CD4 T cells are required to generate protective immunity (8, 33). In these models, the response is mediated by a MHC class I-restricted cellular cytotoxic response (1, 8) and is generated directly by the immunizing gene-modified DCs and not by cross-priming through host APCs (9). These data suggest that adenovirus-mediated endogenous expression of a foreign antigen into DCs leads to epitope presentation through both MHC class I and II. In the current studies, we attempted to determine the role and pathway involved in the CD4 T-cell function when mice are immunized with tumor antigen genemodified syngeneic DCs. Our data strongly support the notion that CD4 T cells are helper cells in this model, which engage the CD40mediated pathway. This helper function cannot be substituted by the supraphysiological production of the type 1 cytokines IL-2, IL-7, or IL-12 when the DCs are transduced with viral vectors expressing levels of these cytokines that are much higher than in CD40-crosslinked DCs. CD40 engagement in the DCs used for immunization leads to detectable changes in cell surface expression of MHC and costimulatory molecules, which may mediate an enhanced ability to stimulate naı¨ve MART-1-specific T cells in the host (34). A consistent finding in our studies is an unexpected high rate of antitumor protection in CD4KO mice immunized with CD40-crosslinked DCs. In four of seven studies, this was superior to AdVMART1/DC immunization in wild-type mice, although this trend was not statistically significant. This trend toward greater protection was not correlated with greater in vitro cytotoxic activity or type 1 cytokine production (in most studies, lysis in 51Cr release assays and number of IFN-␥-producing cells in ELISPOT assays was higher in wild-type mice compared with CD4KO mice immunized with CD40cross-linked DCs). Recent data suggest that CD4 T cells not only provide help to CD8 T-cell activation but also enhance the ability of effector cells to infiltrate tumors and are responsible for maintaining

Fig. 3. Different CD40 cross-linking constructs lead to protective immunity in CD4KO mice. B16 tumor development was monitored in groups of mice immunized with AdVMART1/DC treated ex vivo with trimeric CD40-ligand or anti-CD40 activating antibody (HM40 –3), or mice immunized with AdVMART1/DC s.c. followed by a single i.v. injection of the anti-CD40 activating antibody FGK45.

Fig. 4. CD40 cross-linking led to protective immunity in wild-type C57BL/6 mice where CD4 cells were antibody-depleted. C57BL/6 mice received two i.p. injections of GK-1.5 anti-CD4 antibody before being immunized twice s.c. with AdVMART1/DC with or without CD40 cross-linking using the activating anti-CD40 antibody HM40 –3. Protection from challenge with B16 injected 2 weeks later was monitored. Wild-type AdVMART1/DC versus control, P ⫽ 0.03, versus AdVMART1/DC with CD4 depletion, P ⫽ 0.02, versus AdVMART1/DC-CD40L with CD4 depletion, P ⫽ not significant; control versus AdVMART1/DC-CD40L with CD4 depletion, P ⫽ 0.01. Bars, S.E.M.

the pool of antigen-specific activated CD8 cells conferring antitumor protection (25, 29). Therefore, the finding of a trend toward increased protection but decreased type 1 cytokine production in CD40-ligand immunized CD4KO mice compared with wild-type mice is even more surprising. It is tempting to speculate that, in the absence of CD4 T cells, CD40 ligation leads to stimulatory signals that are not subject to the same negative autoregulatory mechanisms as when T-cell help is mediated by CD4 T cells. When we examined whether the beneficial effects of CD40 crosslinking could enhance antitumor immunity in wild-type mice, we observed variable results. Although overall results suggest a benefit of CD40-mediated DC maturation, clear enhancement of immunity was demonstrated in some but not all replicate experiments. In a similar model, agonistic anti-CD40 antibody treatment of DCs retrovirally transduced to express OVA as a model tumor antigen could not enhance protection to a tumor challenge with B16 cells engineered to express OVA, although these investigators could detect enhanced CTL activity in vitro (35). Because the purity and activation status of DCs cultured in GM-CSF/IL-4 vary considerably between experiments (23), it is possible that some DC preparations may benefit from CD40 cross-linking to enhance immunity in wild-type mice, whereas others may have already received an unrecognized maturation stimulus for optimal immunization in wild-type mice during the DC generation (36). It has also been recognized recently that adenoviral vector transduction leads to efficient DC maturation, resulting in an enhanced antigen-presenting function (37, 38).4 Therefore, adenoviral transduction may be sufficient to provide maturation signals to DCs used to immunize wild-type, CD4-containing mice but may lack a key activation signal to result in protection in CD4KO or CD4-depleted mice. Conversely, this may be a reflection of autoregulatory mechanisms (activation induced cell death, CTLA-4; Refs. 39 – 41), where the immune system may be able to limit overactivation in immunocompetent mice but not in CD4-deficient mice. In a similar model using gp100 gene-modified DCs, CD4 T cells were essential in the immune response, whereas MHC class Irestricted CD8 cells were not required (32). Interestingly, this model also attempted to generate in vivo protection to the murine melanoma cell line B16. We have observed that CD8KO mice in the C57BL/6 background immunized with AdVMART1/DC generate a CD8-independent anti-B16 protective response (data not shown). However, the fact that we can generate an antigen-specific cytotoxic antitumor response in CD4KO by cross-linking CD40 in the DCs and that CD8 4

L. H. Butterfield, personal communication.

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Fig. 5. Engineering DCs to produce type 1 cytokines. A, DCs were transduced with AdVMART1, cotransduced with AdVIL-2, AdVIL-7, or AdVIL-12, and injected into CD4KO mice. Protection to a B16 tumor challenge in CD4KO mice was compared with immunization with AdVMART1/DC ex vivo treated with trimeric CD40-ligand. Wild-type AdVMART1/DC versus control, P ⫽ 0.008; CD4KO AdVMART1/DC-CD40L versus control, versus AdVMART1/DC, versus AdVMART1-AdVIL2/DC, versus AdVMART1-AdVIL7/DC, and versus AdVMART1-AdVIL12/ DC, P ⬍ 0.03 in all cases. B, splenocytes from mice derived from the same groups were harvested and restimulated in vitro for 48 h with a MART-1-expressing irradiated syngeneic cell line. Restimulated cells were assayed for the presence of IFN-␥-producing cells in an ELISPOT assay.

T-cell depletion in CD4KO mice treated with CD40 ligation abrogates protection supports the notion that CD8 cells, and not CD4 T cells, are the effector cells in our model. In summary, CD40 ligation bypasses CD4 T-cell help in murine hosts immunized with MART-1-transduced DCs. The CD40 activation pathway seems to be critical in an environment lacking CD4 cells, but in the presence of CD4 T cells, CD40 engagement is not an absolute requirement for optimal immune activation by adenovirus tumor antigen gene-modified DCs. The mechanism of CD40-mediated bypassing of CD4 help is not mediated by a simple increased production of type 1 cytokines, because DCs engineered to express type 1 cytokines such as IL-2, IL-7, and IL-12 failed to generate protection in CD4KO mice.

Fig. 7. Enhancement of MART-1-specific immunity in C57BL/6 mice. In a series of eight replicate studies, the effect of CD40 antibody ligation was assayed for the ability to enhance AdVMART1/DC-induced protection in immunocompetent wild-type mice. Kaplan-Meier plot of protection from B16 tumor challenges in eight replicate studies where C57BL/6 mice received immunization with AdVMART1/DC-HM40 –3 (n ⫽ 38), AdVMART1/DC (n ⫽ 33), AdVLacZ/DC-HM40 –3 (n ⫽ 24), or unimmunized controls (n ⫽ 40). Comparison of tumor development curves by log-rank test: control versus AdVMART1/DC, AdVLacZ/DC-HM40 –3, and AdVMART1/DC-HM40 –3, P ⫽ 0.001; AdVMART1/DC versus AdVMART1/DC-HM40 –3, P ⫽ 0.067. Comparison by pairwise Cox model: control versus AdVMART1/DC, AdVLacZ/DC-HM40 –3, and AdVMART1/DC-HM40 –3, P ⫽ 0.001; AdVMART1/DC versus AdVMART1/DCHM40 –3, P ⫽ 0.047. Bars, S.E.M.

ACKNOWLEDGMENTS

Fig. 6. Role of CD8 T cells in CD4KO mice immunized with CD40-ligand-treated DCs. CD8 T cells were depleted after DC immunizations in CD4KO mice immunized with AdVMART1/DC-CD40L. CD8 depletion lead to a disappearance of the protection to a B16 tumor challenge noted in the non-CD8-depleted CD4KO mice. Wild-type AdVMART1/DC versus control, P ⫽ 0.01; CD4KO AdVMART1/DC-CD40L versus control, versus AdVMART1/DC, and versus CD8 T cell depleted AdVMART1/DC-CD40L, P ⬍ 0.01 in all cases. Bars, S.E.M.

We thank Dr. Stephen P. Schoenberger for helpful discussions and Kathleen S. Picha and Dr. Elaine K. Thomas (both from Immunex) for the recombinant trimeric murine CD40-ligand and helpful discussions.

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