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Immunology

Enhanced Antitumor Activity Induced by Adoptive T-Cell Transfer and Adjunctive Use of the Histone Deacetylase Inhibitor LAQ824

Dan D. Vo, Robert M. Prins, Jonathan L. Begley, Timothy R. Donahue, Lilah F. Morris, Kevin W. Bruhn, Pilar de la Rocha, Meng-Yin Yang, Stephen Mok, Hermes J. Garban, Noah Craft, James S. Economou, Francesco M. Marincola, Ena Wang and Antoni Ribas
DOI: 10.1158/0008-5472.CAN-09-1456 Published November 2009

Abstract

Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a proapoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 and the adoptive transfer of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: (a) increase in MHC and tumor-associated antigen expression by tumor cells; (b) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and (c) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as a sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein 2, which also showed a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes. [Cancer Res 2009;69(22):8693–9]

Keywords
  • rodent
  • T cells
  • tumor immunity
  • dendritic cells
  • vaccination

Introduction

Metastatic melanoma occasionally responds to immune-based therapies, with durable response rates ranging from 5% to 15% (1, 2). The reasons for these low response rates include suboptimal immune system activation, tumor-derived local immunosuppressive factors, immunosuppressive cells, decreases in tumor antigen and MHC expression, and intrinsic tumor antiapoptotic mechanisms in cancer cells (3). Tumor cell antiapoptotic factors can be pharmacologically modulated, which may lead to improvement in the response rates to tumor immunotherapy (3). Histone deacetylase (HDAC) inhibitors seem particularly suited for this purpose because they have multiple effects on gene transcription and protein function resulting in a proapoptotic phenotype in malignant cells with a favorable therapeutic window compared with normal cells (4–6). In particular, increased histone acetylation induced by HDAC inhibitors results in the activation of genes responsible for generating an intracellular proapoptotic milieu (7–9), increasing surface expression of tumor necrosis factor superfamily death receptors such as Fas, tumor necrosis factor–related apoptosis-inducing receptor, and tumor necrosis factor receptor (8, 10, 11), increased expression of MHC molecules and other molecules involved in antigen processing and presentation (12–15), and increased expression of tumor antigens recognized by CTLs and ligands for NK activating receptors (16, 17).

The class I and II HDAC inhibitor suberoylanilide hydroxamic acid (vorinostat) has been approved by the Food and Drug Administration for the treatment of cutaneous T-cell lymphomas. LAQ824, similar to suberoylanilide hydroxamic acid, is also a pan-HDAC inhibitor hydroxamic acid derivative. LAQ824 has been tested in animal models for its direct antitumor effects mainly against hematopoietic lineage cancer cells (18–20). This agent and a related compound (LHB 589) are in clinical testing in patients with cancer (21). We hypothesized that LAQ824 would render melanoma cells more susceptible to immunotherapy. To test this hypothesis, we developed two models in which LAQ824 was administered together with tumor antigen-specific immunotherapy. Our data show that in vivo treatment with LAQ824 increases the efficacy of antigen-specific immunotherapy in the pmel-1 model and in a vaccine prime-boost model mediated by an improvement in antigen presentation by tumor cells and enhanced function of immune cells, both resulting in increased antitumor activity.

Materials and Methods

Mice, cell lines, and reagents

C57BL/6 (The Jackson Laboratory) and pmel-1 (kind gift from Dr. Nicholas Restifo, Surgery Branch, National Cancer Institute) mice were bred and kept under defined-flora pathogen-free conditions at the American Association for Laboratory Animal Care–approved Animal Facility of the Division of Experimental Radiation Oncology, University of California-Los Angeles, and used under the University of California-Los Angeles Animal Research Committee protocol no. 2004-159. The B16 murine melanoma (American Type Culture Collection) was maintained in DMEM (Mediatech) with 10% FCS (Omega Scientific) and 1% (v/v) penicillin, streptomycin, and amphotericin (Omega Scientific). The HDAC inhibitor LAQ824 was provided by Dr. Peter Atadja (Novartis Institute for Biomedical Research) through a material transfer agreement as a lyophilized powder. LAQ824 was resuspended in 5% tartaric acid and injected i.p. daily at 25 mg/kg for in vivo studies. For in vitro experiments, LAQ824 was resuspended in DMSO at a 10 mmol/L stock solution.

Bone marrow–derived dendritic cells

The generation of dendritic cells from murine bone marrow in cultures of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 was described previously (22). Day 8 dendritic cell cultures were pulsed for 2 h with either gp10025-33 peptide for the pmel-1 adoptive transfer model or TRP2180-188 peptide for the prime-boost vaccine model. Between 5 × 105 and 1 × 106 cells per mouse were injected s.c. in the right flank of mice.

pmel-1 adoptive transfer therapy in vivo model

B16 tumors were implanted s.c. as described previously (22). When tumors reached 5 to 8 mm in diameter, mice received a lymphodepleting regimen of 500 cGy total body irradiation. The following day, 1 × 106 activated pmel-1 splenocytes were adoptive transfer into experimental mice via a lateral tail vein. pmel-1 splenocytes had been activated with 100 units/mL IL-2 (a kind gift from Novartis-Chiron), 10 ng/mL IL-15 (a kind gift from Amgen), and 1 μg/mL gp10025-33 peptide (Beckman Coulter) for 2 to 4 days before i.v. injection. Subsequently, gp10025-33 peptide-pulsed dendritic cells were given s.c. on the day of adoptive transfer and 1 week later, in both cases, followed by 3 days of daily i.p. administration of 500,000 IU IL-2. LAQ824 was given i.p. daily at 25 mg/kg starting on the day of pmel-1 adoptive transfer.

Dendritic cell prime/Listeria monocytogenes–boost animal model

C57BL/6 mice received TRP2180-188 peptide-pulsed dendritic cells prime s.c. followed 1 week later with the L. monocytogenes-TRP2 i.v. boost. Recombinant L. monocytogenes expressing a murine TRP2 transgene were prepared as described previously (23, 24). Mice received s.c. B16 tumor challenge 3 weeks later followed by 25 mg/kg LAQ824 i.p. injection five times weekly.

Flow cytometry analysis

Splenocytes and tumor-infiltrating lymphocytes, obtained from enzymatically digested B16 tumors harvested from mice as described previously (25), were stained with antibodies to CD8αPE (Caltag), CD3PerCP, Thy1.1APC-Cy7, and Thy1.2APC-Cy7 (BD Bioscience), and/or gp10025-33/Db tetramerAPC (Beckman Coulter) and analyzed with a FACSCalibur machine using FCS Express software (DeNovo Software). Intracellular IFN-γ staining was done as described previously (26).

Immunohistochemistry

Immunohistochemical staining was done as described previously (24). Briefly, sections of cryopreserved tissues were incubated with primary antibodies to CD3 (500A2) and CD8 (Ly-2; both from BD Pharmingen) followed by a biotinylated secondary antibody and developed with a 3,3′-diaminobenzidine substrate kit (Vector Labs). Negative controls consisted of isotype-matched rat or hamster IgG in lieu of the primary antibodies listed above.

MTT viability assay

B16 cells, naive C57BL/6 splenocytes, or activated pmel-1 splenocytes were seeded in 96-well flat-bottomed plates (5 × 103 per well) with 100 μL of 10% FCS and incubated for 24 h. Graded dilutions of LAQ824, or DMSO vehicle control, in culture medium were added to each well in triplicate and analyzed following the MTT assay (Sigma).

In vivo cytotoxicity assay

Splenocytes from naive wild-type C57BL/6 mice were pulsed with 50 μg/mL gp10025-33 peptide or the same amount of control OVA257-264 peptide. After 1 h incubation, gp10025-33-pulsed wild-type splenocytes were labeled with and 6 nmol/L CFSE for 10 min at 37°C, whereas control OVA257-264-pulsed splenocytes were differentially labeled with 10-fold dilution of CFSE (0.6 nmol/L). Cells were injected i.v. into experimental mice at 7, 14, 21, and 28 days after pmel-1 adoptive transfer. After 4 h, two mice per group were sacrificed and their spleens were examined for the presence of CFSE-labeled cells. Percent cytotoxic activity was calculated as number of live gp10025-33-pulsed splenocytes divided by the number of live OVA257-264-pulsed splenocytes, which were distinguished based on the 10-fold difference in CSFE fluorescence by flow cytometry.

Gene expression profiling

The study groups are detailed in Supplementary Table S1. Briefly, for in vitro experiments, B16 melanoma and activated pmel-1 cells were incubated with 100 nmol/L LAQ824 or vehicle at 37°C (groups 1 and 2 and groups 7 and 8 in Supplementary Table S1 and Supplementary Fig. S1A and D). After 24 h, cells were collected and resuspended in RNAlater (Applied Biosystems/Ambion) for RNA extraction. For the in vivo experiments, wild-type C56BL/6 mice were injected s.c. with 105 B16 melanoma cells on day 0. On day 10, when the tumors were ∼5 mm in diameter, mice were lymphodepleted with 500 cGy and divided into groups: B16 tumor untreated control or treated with LAQ824 at 25 mg/kg i.p. daily (groups 3 and 4 in Supplementary Table S1 and Supplementary Fig. S1B). The full protocol of pmel-1 adoptive transfer therapy was used alone or with the addition LAQ824; 14 days after pmel-1 adoptive transfer, mice were sacrificed and their spleens (groups 9 and 10 in Supplementary Fig. S1E) and tumors (groups 5 and 6 in Supplementary Fig. S1C) were extracted and immediately fixed for RNA extraction; an aliquot of splenocytes from mice receiving pmel-1 adoptive transfer additionally underwent CD8+ cell purification using magnetic columns (Miltenyi) before RNA isolation. RNA was isolated using Qiagen RNA mini kit. Total RNA was amplified according to previous publications (27, 28) and analyzed in whole-genome murine 36K oligoarrays (Infectious Disease and Immunogenetics Section of Transfusion Medicine, NIH) as described previously (29). Hybridized arrays were scanned on a GenePix 4000 scanner (MDS Analytical Technologies) at variable photomultiplier tube to obtain optimized signal intensities with minimum (<1% spots) intensity saturation.

Statistical analysis

Continuous variables were compared using a paired Student's t test. Survival curves were generated following the Kaplan-Meier method with log-rank test for comparisons from the time from tumor challenge to when mice were sacrificed due to tumors reaching 14 mm in maximum diameter. P values are two-tailed. Resulting data files from the gene expression profiling experiments were analyzed using BRBArrayTools7 and Cluster and TreeView software (GubuSoft; ref. 30).

Results

LAQ824 enhances the antitumor activity of pmel-1 adoptive transfer immunotherapy

Mice with 12-day established s.c. B16 tumors (mean diameter 5-8 mm) received a 500 cGy lymphodepleting dose of total body irradiation and complete pmel-1 adoptive transfer therapy, consisting of 1 × 106 activated pmel-1 T cells adoptively transferred i.v. into B16 tumor-bearing mice along with gp10025-33 peptide-pulsed dendritic cell vaccination and high-dose IL-2 therapy (Fig. 1A). The addition of LAQ824 improved the antitumor activity compared with pmel-1 adoptive transfer therapy alone (Fig. 1B). Combined data from two replicate experiments showed that mice receiving both LAQ824 and pmel-1 adoptive transfer had a statistically significant prolongation in survival (Fig. 1C).

Figure 1.
Figure 1.

Treatment with a HDAC inhibitor enhances the antitumor effects of pmel-1 adoptive transfer therapy. A, experiment timeline. B, tumor growth curves of the mean (SE) tumor diameter of four treatment groups: control mice with lymphodepletion alone, LAQ824 alone after lymphodepletion, complete pmel-1 adoptive transfer therapy, and pmel-1 adoptive transfer therapy together with LAQ824. There were statistically significant differences between the pmel-1 therapy alone compared with the combined therapy. *, P < 0.0001. C, actuarial survival curves of the same four groups of mice pooled from two replicate experiments. LAQ824 treatment significantly prolonged survival of mice receiving pmel-1 adoptive therapy compared with pmel-1 therapy alone. P < 0.0001, log-rank test.

Improvement of antitumor activity in a prime-boost vaccination model with the addition of LAQ824

We tested if LAQ824 could sensitize in a different immunotherapy model. Wild-type C57BL/6 received TRP2180-188 peptide-pulsed dendritic cell prime and recombinant L. monocytogenes-TRP2 boost alone or together with LAQ824 treatment, and mice were then challenged with B16 melanoma (Fig. 2A). Mice given TRP2 prime-boost along with LAQ824 had a statistically significant improvement in tumor protection, with ∼80% of mice remaining tumor-free at the end of the study (Fig. 2B). These results were consistent in two replicate studies. Therefore, the administration of a HDAC inhibitor also improved the antitumor activity in a prophylactic vaccine model against B16 melanoma.

Figure 2.
Figure 2.

Treatment with a HDAC inhibitor improves the antitumor protection to B16 melanoma mediated by a TRP2180-188 peptide-pulsed dendritic cell prime and TRP2 transgenic recombinant L. monocytogenes (rLM) boost. A, experiment timeline. B, LAQ824 improved the survival of mice receiving TRP2-based prime-boost vaccination. Survival was taken from the time of B16 tumor challenge until the tumors reached 14 mm of maximum diameter or at the end of the study. This study was repeated twice with similar results.

Enhanced susceptibility of normal lymphocytes to LAQ824 provides a therapeutic benefit for adoptively transferred T cells

To study the direct effects of LAQ824 exposure, B16 tumor cells, wild-type splenocytes, and activated pmel-1 splenocytes were incubated ex vivo with increasing concentrations of LAQ824 for 2 h and then allowed to rest overnight before being analyzed with a MTT cell viability assay. Activated pmel-1 cells had a significant survival advantage over naive wild-type splenocytes at low concentrations of LAQ824 (1-100 nmol/L), with at least 1 log lower sensitivity (Fig. 3A). Contrary to our expectations, both naive and activated splenocytes were more sensitive to LAQ824-mediated cytotoxicity than B16 when tested in vitro. To test whether a similar survival benefit existed with LAQ824 treatment in vivo, pmel-1 T cells were isolated from C57BL/6 mice after adoptive transfer and the percentage of gp10025-33/Db tetramer-positive cells was quantified over time. Mice treated with pmel-1 adoptive transfer and LAQ824 had a significantly higher percentage and absolute number of gp10025-33/Db tetramer-positive cells in the spleen (Fig. 3B; data not shown), which was consistent for the duration of the experiment (Fig. 3C). We then assessed the ratio of adoptively transferred Thy1.1+ pmel-1 cells to that of endogenous T cells in lymphodepleted, congenic Thy1.2 recipient C57BL/6 mice. In the group of mice treated with both pmel-1 adoptive transfer therapy and LAQ824, the ratio for pmel-1 cells to endogenous T cells was significantly higher than that of mice treated with pmel-1 adoptive transfer alone (Fig. 3D). These in vivo studies confirmed the in vitro results and suggest that one mechanism by which LAQ824 acts is by providing a survival advantage to the adoptively transferred activated T cells.

Figure 3.
Figure 3.

Effects of LAQ824 exposure on the in vitro and in vivo expansion of pmel-1 cells. A, proliferation and cytotoxic activity of LAQ824 against B16 melanoma cells and naive and activated splenocytes in vitro. Cells were exposed to increasing concentrations of LAQ824 for 2 h and analyzed using a MTT cell viability assay. B, gp10025-33/Db tetramer-mediated quantitation of pmel-1 cells recovered from spleens of conditioned C57BL/6 mice on reconstitution with pmel-1 adoptive transfer (AT) with or without addition of LAQ824. The dot plots of flow cytometry correspond to representative mice analyzed on day 7 after adoptive transfer. C, time-course analysis of recovery of gp10025-33/Db tetramer-positive pmel-1 cells at the same conditions as in B, with two mice per group and at different time points after adoptive transfer. D, comparison of the expansion of endogenous Thy1.2 splenocytes and adoptively transferred congenic pmel-1 Thy 1.1+ splenocytes after 500 cGy total body irradiation. Splenocytes were harvested at different time points after adoptive transfer and analyzed by flow cytometry for the two congenic T-cell markers. C and D, gray columns, pmel-1 adoptive transfer therapy alone; black columns, combined therapy with the HDAC inhibitor LAQ824.

Treatment with LAQ824 induces an increased intratumoral infiltration by adoptively transferred pmel-1 cells

Similar experiments were conducted to analyze the intratumoral infiltration by pmel-1 cells. LAQ824 induced a significant increase in both the percentage (Fig. 4A) and the total number of pmel-1 cells per gram of tumor tissue (Fig. 4B) as measured by gp10025-33/Db tetramer staining. Results were corroborated with immunohistochemical staining of CD8+ cells in excised tumors (Fig. 4C and D). The pictures were taken from the tumor periphery to avoid large areas of central necrotic tissue in both groups. Therefore, in vivo treatment with LAQ824 increases B16 intratumoral infiltration by adoptively transferred pmel-1 cells.

Figure 4.
Figure 4.

Increased pmel-1 intratumoral infiltration in mice harboring s.c. B16 melanomas simultaneously treated with pmel-1 adoptive transfer therapy and LAQ824. The percentage (A) and absolute (B) number of gp10025-33/Db tetramer-positive cells were consistently higher in tumors of mice treated with both pmel-1 adoptive transfer therapy and LAQ824 compared with mice treated with pmel-1 adoptive transfer therapy alone. Representative of replicate experiments. Gray columns, pmel-1 adoptive transfer therapy alone; black columns, combined therapy with the HDAC inhibitor (n = 2). *, P < 0.05. Immunohistochemical staining for CD8+ cells in B16 tumor samples of mice treated with pmel-1 adoptive transfer therapy (C) or pmel-1 adoptive transfer therapy in combination with LAQ824 (D). Brown dots, CD8+ cells. Studies were repeated twice with similar results. E, in vivo treatment with LAQ824 in B16 tumor-bearing mice induces expression of the MHC class I alleles H2-Kb and H2-Db as well as intracellular gp100. Single-cell suspensions of B16 tumors were stained immediately after harvesting from mice and analyzed by flow cytometry. Experiments were repeated twice with similar results.

LAQ824 increases expression of MHC class I both in vivo and in vitro

To determine if the HDAC inhibitor increases MHC and/or antigen expression by B16 tumor cells, as has been suggested previously (14, 31), single-cell suspensions of B16 tumor cells from treated and untreated mice were stained for H2-Kb and H2-Db and the gp100 melanoma antigen (HMB45). In two replicate experiments, administration of LAQ824 to B16 tumor-bearing mice induced an increase in expression of MHC class I molecules by surface staining and the gp100 tumor antigen by intracellular staining (Fig. 4E). Therefore, LAQ824 likely increases antigen-driven expansion of pmel-1 cells by increasing MHC and antigen expression.

LAQ824 enhances the functional activity of adoptively transferred pmel-1 cells in vivo

We then analyzed the effects of LAQ824 on the ex vivo effector functions of pmel-1 cells. pmel-1 cells were restimulated in vitro for 6 h with the cognate gp10025-33 peptide, compared with an irrelevant control peptide (AH1), in low concentrations of IL-2. IFN-γ was measured by intracellular cytokine staining. Splenocytes from mice that had received the combined therapy had higher production of IFN-γ when stimulated ex vivo with cognate gp10025-33 peptide (Fig. 5A). These findings were reproducible among replicate mice and specific for the gp10025-33 peptide (Fig. 5B). We then tested the cytotoxic activity of adoptively transferred pmel-1 cells with or without the addition of LAQ824 treatment in a CFSE-based in vivo cytotoxicity assay (32). As targets, wild-type splenocytes were pulsed with either control OVA257-264 or the cognate gp10025-33 peptide and differentially stained with CFSE before being injected into mice that had previously been given pmel-1 adoptive transfer. pmel-1–reconstituted mice treated with LAQ824 showed increased cytotoxic activity against gp10025-33 peptide-pulsed splenocytes compared with mice receiving pmel-1 adoptive transfer alone, although there was no significant killing of splenocytes pulsed with OVA257-264 control peptide (Fig. 5C). The results were consistent in assays repeated weekly after pmel-1 adoptive transfer until study day 28 (Fig. 5D). Taken together, these experiments suggest to us that this HDAC inhibitor provides a functional benefit to pmel-1 cells shown by increased antigen-specific cytokine production and cytotoxic activity in vivo. Alternatively, the preferential depletion of naive endogenous lymphocytes may increase the ratio of pmel-1 effector cells leading to an apparent improvement in functionality.

Figure 5.
Figure 5.

LAQ824 functionally enhances adoptively transferred pmel-1 cells. A, T-cell activation measured by intracellular IFN-γ staining of pmel-1 splenocytes on ex vivo stimulation with the cognate gp10025-33 peptide. Dot plots depict representative samples at day 28. B, antigen-specific intracellular IFN-γ release shown after splenocytes from pmel-1 reconstituted mice, with or without additional LAQ824 treatment, were stimulated with IL-2 alone, IL-2 plus AH.1 irrelevant peptide, or IL-2 plus cognate gp10025-33 peptide for 6 h (day 28). C, representative flow cytometry histograms of in vivo cytotoxicity of OVA257-264 and gp10025-33 peptide-pulsed and CFSE-labeled syngeneic splenocytes in mice 21 d after pmel-1 adoptive transfer therapy with or without addition of LAQ824. Percent lysis was calculated comparing the number of events positive for the fluorescence derived from the low CFSE-labeled control OVA257-264-pulsed splenocytes (left) with the number of events from the high CFSE-labeled gp10025-33-pulsed splenocytes (right). D, combined results of two mice per group with in vivo cytotoxicity assays conducted at four different time points after pmel-1 adoptive transfer. Bar graphs representing the percentage of gp10025-33/Db tetramer-positive cells (E) as well as IFN-γ–producing CD8+ cells on gp10025-33 peptide stimulation (F) analyzed in mice that had never been challenged with B16 melanoma and with splenocytes obtained at different time points after pmel-1 adoptive transfer therapy and while on in vivo therapy with LAQ824. B and D-F, representative results of two mice analyzed in replicate experiments. Gray columns, pmel-1 adoptive therapy alone; black columns, combined therapy with LAQ824. *, P ≤ 0.05.

LAQ824 effect on adoptively transferred pmel-1 cells is independent of B16 tumor presence

To further determine if HDAC inhibitor treatment had direct effects on immune effector cells or its effects were indirectly mediated by HDAC inhibitor-modulated B16 cells, we tested the effects of LAQ824 when administered to non–tumor-bearing mice reconstituted with pmel-1 adoptive transfer. In the absence of gp100 antigen presented by B16 tumor cells, the addition of LAQ824 to pmel-1 adoptive transfer still resulted in a significantly increased percentage of gp10025-33/Db tetramer-positive cells (Fig. 5E) as well as IFN-γ–producing CD8+ cells at each time point evaluated (Fig. 5F). Therefore, the HDAC inhibitor benefits antigen-specific immune effector cells independent of antigen presentation by the tumor.

Gene expression profiling confirmed the modulation of antigen, MHC, and immune gene expression by HDAC inhibitor therapy

In an effort to identify gene targets modulated by the combination of pmel-1 adoptive transfer and HDAC inhibitor treatment, we performed whole-genome expression profiling comparing untreated and LAQ824-treated B16 and pmel-1 samples both in vitro and in vivo (study groups are detailed in Supplementary Table S1). The whole-genome profiling was analyzed using unsupervised clustering, and the replicate samples from each of the 10 groups clustered together (data not shown). To eliminate tissue or sample type-specific gene signatures, we compared LAQ824-treated samples versus control samples directly. We noted marked differences in gene expression profiling B16 and pmel-1 cells exposed in vitro or in vivo to LAQ824 (Supplementary Fig. S1). A consistent finding in the in vivo experiments was that silver (the gene denomination of the gp100 melanoma tumor antigen) was one of the genes with the most significant decrease in B16 tumors (likely containing tumor cells, immune cells, and stromal cells) harvested from mice that had received the complete pmel-1 adoptive transfer with LAQ824 (Supplementary Fig. S1). It was not restricted to this tumor-associated antigen because expression of other melanosomal antigens, such as the murine homologue of Melan-A, also decreased after combined therapy. Given that these expression data were taken from late time points (2 weeks after pmel-1 adoptive transfer), the results strongly suggest that strong, targeted-specific elimination of tumor-associated antigen occurred in B16 melanomas in vivo when combined with HDAC inhibitor treatment.

Multiple lines of evidence, including our flow cytometry studies, suggest that HDAC inhibitors modulate MHC molecules and antigen processing machinery. Most MHC molecules selected from differentially expressed transcripts had low expression in vitro regardless of LAQ824 exposure but high expression in the in vivo harvested samples only after pmel-1 adoptive transfer therapy (Supplementary Fig. S1B compared with Supplementary Fig. S1C). This gene expression pattern followed closely the gene expression pattern of IFN-related genes such as IFN-inducible GTPase 1 and 2, IFN-γ–induced GTPase, and IFN-regulatory factor 1; other immune cytokines and signaling genes such as signal transducer and activator of transcription 1 and 2, Janus kinase 2, IL-33, and granzyme B; other immune activating genes such as T-cell receptor-β, Fc receptors, and CD52; and chemokines and their receptors such as chemokine (C-C motif) ligands 5, 7 to 9, and 12. From these studies, we concluded that the effects of LAQ824 on the different cell types that contribute to this combined therapy are markedly different depending on the analysis context, with a final overall effect of favoring an effective antitumor response when analyzing samples treated in vivo with LAQ824.

Discussion

In these studies, we show that the HDAC inhibitor LAQ824 improves the antitumor activity of two tumor immunotherapy strategies in the B16 murine melanoma model. In the pmel-1 adoptive transfer model, we defined three main mechanisms of this beneficial effect. One is an increase in MHC and antigen expression by tumor cells, which was shown when tumors were treated with single-agent LAQ824 therapy in vivo. The combined adoptive transfer pmel-1 and HDAC inhibitor therapy resulted in markedly decreased expression of gp100 and other tumor-associated antigen expression, which we have interpreted as representing tumor cell killing. However, the effects on antigen presentation alone are unlikely to explain the beneficial effects of the combined therapy, because agents such as IFN-γ induce increases in MHC class I and antigen expression at much higher levels than this HDAC inhibitor (33). A second mechanism is a decrease in competing endogenous lymphocytes in recipient mice, resulting in a selective advantage for the repopulation with adoptive transfer pmel-1 cells. Our in vivo studies confirmed the results of in vitro differential sensitivity in MTT assays and showed a selective repopulation of lymphodepleted mice with pmel-1 cells compared with congenic endogenous lymphocytes. These results suggested to us that the effects of LAQ824 may work both by enhancing antigen-driven T-cell expansion through increased tumor-associated antigen presentation by tumor cells in vivo and by enhancing homeostatic proliferation mediated by the differential sensitivity of naive recipient splenocytes and the relative resistance of the adoptive transfer activated pmel-1 cells. This hypothesis is further supported by a comparable pmel-1 expansion in both the presence and the absence of B16 tumors in recipient mice. A third mechanism is an improvement in the functional activity of the adoptively transferred antitumor CTLs. We acknowledge that the data could also be interpreted as a quantitative increase pmel-1 cells in the combined therapy groups without increase in functionality. The interpretation is supported by the high mean fluorescence intensity for IFN-γ intracellular cytokine staining in the combined therapy group when restimulated with cognate peptide as well as increased lytic activity in vivo, suggesting increased functionality in addition to an absolute increase in number.

There is a body of recent literature suggesting that HDAC inhibitors have immunosuppressive effects (34, 35), which includes seemingly discording results of effects on T regulatory cells (36, 37). However, our data add to the previously reported findings of HDAC inhibitor-induced immune potentiation, including the MS-275 increasing the antitumor activity of high-dose IL-2 against the Renca murine kidney cancer model (36), the HDAC inhibitor trichostatin A against the modified lung cancer cell line TC-1 (15), and the HDAC inhibitor depsipeptide (FK228) also against B16 melanoma as in our experience (31). Therefore, it is likely that the immunomodulating effects of HDAC inhibitors on T regulatory cells and other immunosuppressive cell subsets are not dominant in the setting of powerful tumor immunotherapy strategies.

A major benefit of the gene expression profiling in this model is to provide evidence that the effects of the HDAC inhibitor treatment in vitro had markedly different effects compared with the analysis of gene expression profiling in the same cell subsets (pmel-1 and B16) when analyzed in vivo. Comparison of the gene expression patterns in vitro and in vivo suggested to us that the effects of HDAC inhibitor therapy was likely a combined effect that includes interactions between the adoptively transferred cells, the tumor microenvironment and the tumor cells, as opposed to a single effect in one of these cell subsets.

In conclusion, our studies provide insights on how a pan-HDAC inhibitor can improve vaccine-based and adoptive cell therapy-based immunotherapies. Given the reproducibility of our findings in two different tumor immunotherapy models as well as data from others (15, 31, 36), we believe that HDAC inhibitors of the same class as LAQ824, such as the Food and Drug Administration–approved vorinostat or the related compound LBH 589 currently in clinical testing, could improve tumor immunotherapy in translational clinical studies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Grant support: Harry J. Lloyd Charitable Trust, P50 CA086306, Waxman Foundation, W.M. Keck Foundation, and Monkarsh fund. Flow cytometry was done at the University of California-Los Angeles Jonsson Comprehensive Cancer Center Core Facility, which is supported by the NIH award CA16042.

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.

We thank Dr. Peter Atadja (Novartis) for providing LAQ824 and the gift of IL-2 (Novartis) and IL-15 (Amgen) and Caius G. Radu, M.D., Benjamin Bonavida Ph.D. and Siavash K. Kurdistani, M.D. (all at University of California-Los Angeles) for helpful discussions.

Footnotes

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Cancer Research: 69 (22)
November 2009
Volume 69, Issue 22

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Enhanced Antitumor Activity Induced by Adoptive T-Cell Transfer and Adjunctive Use of the Histone Deacetylase Inhibitor LAQ824
Dan D. Vo, Robert M. Prins, Jonathan L. Begley, Timothy R. Donahue, Lilah F. Morris, Kevin W. Bruhn, Pilar de la Rocha, Meng-Yin Yang, Stephen Mok, Hermes J. Garban, Noah Craft, James S. Economou, Francesco M. Marincola, Ena Wang and Antoni Ribas
Cancer Res November 15 2009 (69) (22) 8693-8699; DOI: 10.1158/0008-5472.CAN-09-1456
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