An epigenetic vaccine model active in the prevention and treatment of melanoma

Mouse B16 melanoma, EL4 thymoma cell lines and hybridoma GK1.5, 2.43 and 53-6.7 cells (ATCC, Manassas, VA) were maintained in culture as specified. Six to eight week old female C57BL/6 (B6) (NCI, Bethesda, MD), MHC class I deficient B6.129-β₂m^tm1 [class I^-/-], class II deficient B6.129-H2^dlAb1-Ea (class II^-/-) (Jackson Laboratory, Bar Harbor, ME), class I and class II double deficient B6.129-Aββ^tm1-β₂m^tm1 (class I^-/--II^-/-) (Taconic, Germantown, NY) and ovalbumin-specific I-A^b-restricted TCR transgenic (OT-II) (Protul Shrikant, RPCI, Buffalo, NY) mice were maintained in the Department of Laboratory Animal Resources at RPCI. Principles of laboratory animal care (NIH publication 85-23, revised 1986) were followed and all work was carried out under RPCI IACUC approval. TSA (Wako Biochemical, Richmond, VA), H₂O₂ (Sigma, St. Louis, MO) and mouse IL-2 (R&D System, Minneapolis, MN) were diluted in ethanol, water and PBS containing 0.5% BSA, respectively.

R-Phycoerythrin conjugated anti-mouse I-A^b, H-2D^b, CD4, CD40, CD80, CD86 and Pan-NK (DX5), FITC conjugated anti-mouse I-A^b and CD3, PE-Cy5.5 conjugated anti-mouse CD8 and CD11c mAb, isotype controls matched to each antibody (Pharmingen, San Diego, CA) and FITC conjugated annexinV (Caltag, Burlingame, CA) were used in flow cytometry experiments as previously described [4]. The gated cell populations included both apoptotic and non-apoptotic cells. In all preparations utilized here, the adherent cells studied were >95% viable by trypan blue exclusion.

TSA-treated or untreated B16 cells (1 × 10⁵ trypan blue negative adherent cells) were injected s.c. in the ventral trunk of the mice. This tumor challenge dose is 10 fold higher than the minimum number of untreated cells required to generate palpable tumor in 100% of mice within 3 weeks after injection. Treated cells were assayed prior to injection to ensure consistency between experiments in MHC and costimulatory molecule expression as well as apoptosis. Tumors were measured every 3 days and mice were euthanized when tumor diameter reached 1 cm. After 40 days, all tumor-free mice were re-challenged s.c. with untreated B16 (1 × 10⁵) cells in the opposite site and observed for another 60 days. In addition to palpable tumor measurement, the absence of evidence of tumor in immune mice was confirmed by visual inspection. At the end of the study period (100 days after vaccination), a mouse from each group of tumor-free mice was euthanized and the tumor inoculation sites and regional lymph nodes dissected and examined for evidence of tumor (no visible tumor nodule or other evidence of tissue aberration) and compared with a palpable tumor-bearing mouse (visible tumor nodules in lymph nodes). Some tumor-free B6 mice were re-challenged with EL4 (1 × 10⁴) cells simultaneously as a control for tumor specificity. For the treatment model, B6 mice bearing palpable (~0.5 mm) B16 tumor, 5 days after s.c implantation in the trunk, were vaccinated with TSA- treated and irradiated (2000 Gy) B16 cells in the opposite side. Groups of tumor-bearing control mice received irradiated B16 cells or were left untreated. Mice that became tumor-free were re-challenged 42 days latter with wild type B16 in the trunk and observed for another 40 days.

To determine tumor immunity, tumor-free mice, 30 days after vaccination with TSA-treated B16 cells, were re-challenged with untreated B16 cells and observed for an additional 15 days. Spleens isolated from three immune or control mice were disrupted and splenocytes were purified over nylon wool (Polyscience, Warrington, PA). Non-adherent lymphocytes (5 × 10⁵) were re-stimulated with TSA-treated (500 nM for 48 h) and irradiated (200 Gy) tumor cells (2.5 × 10⁵) in RPMI-1640 with IL-2 (10 U/ml). After 4 days, T-cell-enriched (>90%) viable cells were isolated after centrifugation through Ficoll-Paque and the level of anti-B16 cytotoxicity was determined utilizing a standard 4.5 h ⁵¹Cr-release assay [4].

The mouse IFN-γ ELISpot kit (BD Bioscience, San Diego, CA) was used to determine antigen specific IFN-γ-secreting cells in spleens of immune mice as described [19]. Briefly, RBC-depleted splenocytes (1 × 10⁶ cells/well) isolated from immune and control mice were incubated in triplicate wells with B16 tumor cell lysate (~8 × 10⁴ cell lysate/well) or 5 μM mgp100_25–33 peptide (Invitrogen, Grand Island, NY) for 24 h. IFN-γ spots were developed with AEC substrate and counted using a Zeiss Imaging system. B6 mice bearing palpable tumor 15 days after inoculation of untreated B16 (1 × 10⁵ cells) and naïve mice were used as controls. Splenocytes (2 × 10⁵ cells/well) from naïve mice treated with PMA (40 ng/ml) and Ionomycin (1 μM) served as positive control. For in vitro antigen presentation assays, popliteal lymph node T cells (2 × 10⁵), isolated from ovalbumin-primed OT-II mice and purified magnetically (Pan T cell isolation kit, Miltenyi, Auburn, CA), were assayed in triplicate with TSA-treated (500 nM for 48 h) or untreated B16 cells (1 × 10⁵) for 24 h. Splenocytes isolated from OT-II mice were used as control APCs. Tumor cells and splenocytes were pulsed with ova-peptide_322–338 or the control E333A ova-peptide_322–338 (10 μM) and irradiated (2000 Gy and 30 Gy respectively) before use in the ELISpot assays to measure IFN-γ-secreting T cells.

Anti-CD4 (GK1.5) and CD8 (2.43) hybridomas were grown i.p. in SCID mice and ascites fluid was partially purified by ammonium sulfate precipitation. To deplete CD4⁺ or CD8⁺ T cells, B6 mice were injected i.p. with CD4 or CD8 mAb (200 μg in 200 μl PBS/mouse), given at day -2, 0, +2, +4, +8 and +14. Rabbit anti-asialoGM1 γ-globulin (Wako), reconstituted in water and diluted in PBS, was used for NK cell depletion using the same schedule. Control mice received purified rat IgG (Chemicon International, Temecula, CA). Control and depleted mice were vaccinated with TSA-treated B16 cells after the 2^nd dose of antibody injection and observed for 30 days. Depletion was assessed on the day of vaccination and a week later by flow cytometric analysis of splenocytes for CD3⁺CD4⁺, CD3⁺CD8⁺ T cells and DX5⁺ NK cells.

Bone marrow (BM) cells were harvested from femur and tibia of groups of donor (B6 and class I^-/--II^-/-) mice by flushing the bones with RPMI-1640. RBCs were lysed using Tris-buffered ammonium chloride and single cell suspensions were obtained by passing BM cells through a cell strainer [20]. T cells were depleted from BM preparations by incubation with CD8 (53-6.7) and CD4 (GK 1.5) mAb (1:10 dilution) for 60 min at 4°C, followed by lysis with low-tox-M rabbit complement (Cederlane, Ontario, Canada) for 90 min at 37°C in 5% CO₂ incubator [21]. In preliminary experiments, BM cells after treatment with hybridoma supernatants (53-6.7 or GK1.5) were analyzed by flow cytometry using CD3, CD4 (L3T4) and CD8 (5H10) mAb to confirm the depletion of CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells. One day before BM infusion, recipient B6 mice were injected i.p. with a single dose anti-asialoGM1 (20 μl in 200 μl sterile PBS) antibody to prevent NK mediated rejection of BM. The recipient mice were exposed to 11 Gy total body irradiation (TBI) administered in two treatments 3 h apart from a ¹³⁷Cs-radiation source on the day of BM infusion. A total of 5 × 10⁶ BM cells from donor mice were injected into the tail vein of each recipient. Chimeras were provided with water containing 2 mg/ml neomycin sulfate for 4 weeks after irradiation. Splenocytes, isolated from irradiated mice 2 days after TBI, were analyzed by flow cytometry to confirm the elimination of CD11c⁺ APCs and CD3⁺ T cells and >99% elimination was observed. To obtain a dendritic cell (DC) enriched APC population, low-density splenocytes were isolated by collagenase digestion [22]. At 4–8 weeks after BM transplant, chimerism was evaluated using PBLs and splenocytes isolated from chimeric mice.

Apoptotic B16 cells were isolated using annexinV microbead kit, MS separation column and magnetic separator (Miltenyi, CA) according to the manufacturer's protocol. Briefly, TSA or H₂O₂-treated B16 cells were labeled with annexinV microbead for 15 min at 6–12°C and passed through a separation column in a magnetic field. AnnexinV positive (an⁺) apoptotic cells were retained in the column while annexinV negative (an^-) non-apoptotic cells ran through. Apoptotic cells were eluted after removal of the column from the magnetic field.

Previously we showed that TSA treatment elicits the expression of MHC class II and costimulatory molecules on the mouse plasma cell tumor J558 and that vaccination with epigenetically altered tumor cells generates tumor-specific immunity [4]. To extend this to another tumor and to further analyze the mechanisms involved in immunity generation we used the MHC class II negative B16 melanoma. To determine the cell surface expression of MHC class I, class II, CD40, CD80 and CD86 in relation to apoptosis induction, TSA-treated (50 nM-1 μM, 12–48 h) adherent (~95% viable) B16 cells were analyzed by flow cytometry. The conditions inducing maximal expression are presented in Figure 1A. This Figure demonstrates enhancement of the expression of MHC class II, CD40 and CD86 on B16 cells at 24 h and 48 h of TSA treatment. TSA treatment also slightly enhanced the low-level expression of class I found on untreated cells. Treatment with 500 nM TSA for 48 h, a condition that produced 46% an⁺ cells in the adherent B16 population, in addition to class II, CD40 and CD86 induced low-level expression of CD80. TSA-treated B16 cells retained enhanced expression of these genes over 24 h after withdrawal of treatment in vitro (data not shown). These results demonstrate that TSA treatment induces apoptosis and elicits expression of class II and costimulatory molecules on B16 tumor cells similar to the results previously reported on the J558 plasmacytoma [4].

To determine whether TSA treatment altered tumorigenicity and immunogenicity, B16 cells treated with different concentrations of TSA were inoculated into mice. As shown in Table 1, nearly all control and 90% of the 250 nM TSA treated B16 (~20% an⁺ cells) injected mice developed tumors. Using 500 nM TSA (24 h) treated B16 (~40% an⁺ cells), 30% of the mice injected were tumor-free for more than 40 days. However, when mice were inoculated with B16 cells treated with 500 nM TSA for 48 h (~50% an⁺ cells), 80% of the mice were tumor-free after 10 days, by which time all controls had developed tumors, and more than 60% of the mice remained tumor-free for over 40 days (Table 1 and Figure 1B). When tumor-free immune mice were re-challenged with untreated B16, 100% of the TSA-treated B16 vaccinated mice showed lasting immunity (Figure 1C). The immunity generated by TSA-treated B16 vaccination is tumor specific as the unrelated EL4 thymoma developed tumor in 100% of the immune mice (data not shown). In similar experiments, B16 cells treated with high dose (750 nM for 48 h) TSA, containing ~90% an⁺ cells, demonstrated immunity in 40% of the mice after vaccination (data not shown). This high dose was associated with greater number of an⁺ cells did not improve the effectiveness of the vaccine. These results indicate that, similar to the TSA-treated plasmacytoma vaccine, TSA-treated melanoma cells generate immunity and preparations containing approximately 50% an⁺ cells were most effective in inducing tumor specific lasting immunity [4].

To determine whether CTLs were elicited in immune mice, T-cell-enriched splenic lymphocytes were isolated from TSA-treated B16-vaccinated mice that remained tumor-free after re-challenge and assessed for their ability to lyse untreated B16 cells. As shown in Figure 2A, splenic lymphocytes derived from immune mice displayed substantial B16 lytic activity (26%) compared with splenocytes derived from control mice (<2%) at an effector: target ratio 50:1. The cytotoxic lymphocytes induced by TSA-treated melanoma vaccination were tumor specific, since lysis of the unrelated target EL4 (1%) was significantly lower than that of B16 (Figure 2A).

ELISPOT assays were employed to measure IFN-γ production by cytotoxic lymphocytes from immune mice. As shown in Figure 2B, splenocytes derived from mice immunized with TSA-treated B16 and challenged with untreated tumor cells showed significant increases in the numbers of IFN-γ-producing cells after B16 cell lysate or mgp100_25–33 peptide stimulation compared to splenocytes derived from naive mice. The number of IFN-γ-producing cells found in splenocytes isolated from B16 tumor-bearing mice was significantly lower than the immune mice. PMA/Ionomycin treatment induced a high number of IFN-γ-producing cells in splenocytes isolated from naïve, tumor-bearing and immune mice. The presence of cytotoxic and IFN-γ-producing cells in the spleens of immune mice, together with the demonstration of long-term immunity after vaccination with TSA-treated B16 cells, suggest that epigenetically altered tumor cells are capable of inducing cell mediated tumor specific effectors and immunity.

To determine the role of CD8⁺ and CD4⁺ T cells in immunity, MHC double knockout class I^-/--II^-/- and single knockout class I^-/- or class II^-/-mice were used in tumorigenesis experiments, since these MHC disrupted mice lack the related subset of mature T cells (data not shown) [23]. The incidence of tumor rejection in class I^-/--II^-/-, class I^-/- and class II^-/- mice (Figure 2C-11%, 11%, 31% tumor-free respectively) 40 days after TSA-treated B16 inoculation, is significantly lower than in immunocompetent B6 mice (73% tumor-free). Although, the percentage of tumor-free mice in the class II^-/- group is numerically higher than the class I^-/- group, the difference was not statistically significant (p = 0.08). In subsequent re-challenge with untreated B16, all the MHC deficient mice developed tumors (data not shown) while immunocompetent mice remained tumor-free.

To more specifically evaluate the contribution of T cell subsets to protective immunity generated by TSA-treated tumor cell vaccination, in vivo depletion was performed. Calculation of the total number of CD4⁺ and CD8⁺ T cells in each spleen, isolated from the respective T cell depleted mice, compared to undepleted control spleens, demonstrated >95% depletion of specific subsets (data not shown). Figure 2D shows that the percentage of tumor-free mice in CD8⁺ or CD4⁺ T cell depleted groups (12.5% and 37.5%, respectively), 3 weeks after vaccination with TSA-treated B16 cells, is significantly lower than the control groups (~75%). NK cells can be responsible for tumor rejection by direct lysis of tumor cells and by producing cytokines that recruit and activate DCs and T cells [24, 25]. Depletion of NK cells prior to vaccination, as shown in Figure 2D, completely abrogated the anti-tumor effect of TSA-treated B16 vaccination. These results demonstrate that CD4⁺, CD8⁺ T cells and NK cells are all required for generation of the immune response by TSA-treated B16 cells and that NK cells are particularly important in tumor rejection. However, the TSA-treated B16 tumorigenesis data from T cell deficient class I^-/--II^-/- mice and our previous studies in SCID mice with TSA-treated plasmacytoma [4] suggest that NK cells alone, in the absence of T cells, are not sufficient to induce effective anti-tumor immunity in these epigenetic vaccine models.

In order to determine the mechanism of epigenetic vaccine induced immunity, we further explored whether B16 melanoma cells are converted to APC following TSA treatment. Figure 3A demonstrates that TSA-treated B16 cells can present ova-peptide_322–338 to OT-II T cells in the context of MHC class II in vitro. In view of these studies and to determine the role of direct antigen presentation in elicitation of the immune response by epigenetic tumor cell vaccination in vivo, we developed MHC double knockout BM (DKO-C) chimeras using class I^-/--II^-/- donors and irradiated B6 recipients. Control BM (B6-C) chimeras were produced using immunocompetent B6 donors and irradiated B6 recipients. Both class I and class II expressing APCs were significantly reduced in DKO-C chimeras and they were repopulated with CD4⁺ and CD8⁺ T cells (see Additional file 1). Since MHC class I and/or class II mediated antigen presentation by BM derived APCs initiates cross priming of T cells [1], the DKO-C chimeras are expected to have lost the ability to present antigen from cross priming. B6-C chimeras did not show a deficiency in APC MHC expression, were re-populated with T cells (see Additional file 1) and were expected to have intact cross-presentation capability from both MHC pathways. When these chimeras were inoculated with TSA-treated or untreated B16 cells, 50% of control chimeras and 37.5% of DKO-C chimeras remained tumor-free 10 days after inoculation of TSA-treated B16 cells while all the B6-C and DKO-C chimeras had developed untreated B16 tumors (Figure 3B). Delayed tumor generation, at day 10, in B6-C (p <0.01) or DKO-C chimeras (p <0.03) after inoculation of TSA-treated cells was statistically significant compared to B6-C or DKO-C chimeras injected with untreated B16. These data demonstrate that, in the absence of cross-presentation, tumor generation is delayed suggesting that direct antigen presentation by TSA-treated tumor cells may contribute to delayed tumor growth. However, neither control nor MHC deficient chimeras were able to provide the long-term protection after vaccination with TSA-treated B16 cells that was demonstrated by the immunocompetent mice (Figure 1). This observation also suggests that the BM chimeras used for these experiments, although repopulated with T cells, may not be completely immunocompetent as has been noted by others [26]. Additionally, a recent report suggests that direct presentation by TSA-treated B16 cells may generate partial activation of T cell effector function, but is unable to provide long-term protection in the absence of other signals [27]. This may, at least in part, explain the ability in our chimeric experiments to elicit short-term but not long-term immunity.

The tumorigenesis studies with B16 cells (Table 1) showed that the highest level of immunity was obtained using vaccine preparations containing apoptotic cells. This raises the possibility that TSA treatment enhances immunity, at least in part, by induction of apoptosis. To further elucidate the role of apoptotic cells in the B16 model and the ratio of apoptotic to viable cells required for optimal immunity, populations of apoptotic and non-apoptotic adherent B16 cells were separated using annexinV magnetic beads. We also compared the effects of TSA-apoptotic cells with those elicited by H₂O₂, a potent inducer of apoptosis via the oxidative pathway [28]. The dose of H₂O₂ was determined by titration of B16 cells with H₂O₂ at various concentrations and incubation times with apoptosis measured by flow cytometry. Flow cytometric analysis of the separated TSA or H₂O₂-treated B16 populations showed that >99% of the cells in the apoptotic fraction were an⁺ and >85% cells in the non-apoptotic fraction were an^- (data not shown). To determine tumorigenicity and immunity, groups of mice were inoculated with TSA or H₂O₂-treated apoptotic cells, TSA-treated non-apoptotic or mixed (50% TSA-treated apoptotic + 50% TSA-treated non-apoptotic) B16 cells. As shown in Figure 4A, 40% of mice that received mixed B16 inocula were tumor-free at day 20, while all the mice injected with TSA-treated non-apoptotic or untreated B16 cells developed tumors. TSA or H₂O₂-treated apoptotic cells did not replicate in vitro (data not shown) and did not produce tumors in vivo (Figure 4A). To determine the level of immunity induced, all tumor-free mice were challenged after 3 weeks with untreated B16 cells. The TSA-treated live plus apoptotic 50:50 mixture generated a significantly higher level of durable immunity (Figure 4B, 88% tumor-free) compared to the TSA-treated apoptotic inoculums (30% tumor-free) and H₂O₂-treated apoptotic cells failed to elicit long-term immunity. The lasting immunity induced by TSA-treated apoptotic cells is statistically significant compared to H₂O₂-treated apoptotic cells and untreated controls (p <0.04). These data suggest that TSA-treated apoptotic cells may play a role in immunity and TSA-treated preparations containing ~50% apoptotic cells, in addition to non-apoptotic cells, are most effective in inducing long-term immunity. Importantly, these data also suggest that generation of durable immunity by annexinV positive tumor cells may vary depending on the apoptosis-inducing agents [28].

To evaluate the potential of clinical translation of the TSA-treated vaccine model, two sets of tumorigenesis experiments were performed. Firstly, to eliminate tumor growth from the vaccine inocula, we lethally irradiated tumor cells before inoculation and found that 100% of the mice injected with TSA-treated or untreated B16 cells (irradiated) did not develop tumor (data not shown). After 3 weeks, tumor-free mice from both groups were challenged with wild type B16; 50% of the TSA-radiation group and only 12.5% of the control-radiation group showed durable immunity (Figure 5A). These data suggest that killing the TSA-treated tumor cells with radiation does not reduce the vaccine efficacy. Additionally, to determine the efficacy of the irradiated vaccine in a therapeutic model, palpable B16 tumor-bearing mice were inoculated with TSA-treated or untreated B16 cells (irradiated) on the opposite side. As shown in Figure 5B and 5C, 100% of mice receiving a single injection of TSA-treated (irradiated) B16 vaccine were tumor-free at 42 days and 50% of them remained tumor-free 40 days after wild type tumor challenge. In 75% of tumor-bearing mice, the tumor regressed after injection with irradiated B16 cells (Figure 5B), but none of them were protected against challenge with wild type B16 (Figure 5C). These data demonstrate that vaccination with TSA-treated (irradiated) tumor cells can suppress primary tumor growth as well as promote long-term immunity in this treatment model.