Skip to main content
NCBI home page
Molecular Therapy logoLink to Molecular Therapy
. 2011 Sep 20;20(2):432–442. doi: 10.1038/mt.2011.183

Genetic Targeting of the Active Transcription Factor XBP1s to Dendritic Cells Potentiates Vaccine-induced Prophylactic and Therapeutic Antitumor Immunity

Shenghe Tian 1, Zuqiang Liu 1, Cara Donahue 1, Louis D Falo Jr 1,2, Zhaoyang You 1–,3
PMCID: PMC3277233  PMID: 21934655

Abstract

In vivo dendritic cells (DC) targeting is an attractive approach with potential advantages in vaccine efficacy, cost, and availability. Identification of molecular adjuvants to in vivo “modulate ” DC to coordinately render improved Th1 and CD8 T cell immunity, and attenuated deleterious Treg effects, is a critical challenge. Here, we report that in vivo genetic targeting of the active transcription factor XBP1s to DC (XBP1s/DC) potentiated vaccine-induced prophylactic and therapeutic antitumor immunity in multiple tumor models. This immunization strategy is based on a genetic vaccine encoding both cytomegalovirus (CMV)-driven vaccine Aghsp70 and DC-specific CD11c-driven XBP1s. The novel targeted vaccine induced durable Th1 and CD8 T cell responses to poorly immunogenic self/tumor antigen (Ag) and attenuated tumor-associated Treg suppressive function. Bone marrow (BM)-derived DC genetically modified to simultaneously overexpress XBP1s and express Aghsp70 upregulated CD40, CD70, CD86, interleukin (IL)-15, IL-15Rα, and CCR7 expression, and increased IL-6, IL-12, and tumor necrosis factor (TNF)-α production in vitro. XBP1s/DC elevated functional DEC205+CD8α+DC in the draining lymph nodes (DLN). The data suggest a novel role for XBP1s in modulating DC to potentiate tumor vaccine efficacy via overcoming two major obstacles to tumor vaccines (i.e., T cell hyporesponsiveness against poorly immunologic self/tumor Ag and tumor-associated Treg-mediated suppression) and improving DEC205+CD8α+DC.

Introduction

Given the importance of dendritic cells (DC) in inducing, regulating, and maintaining antigen (Ag)-specific T cell immunity, numerous ex vivo DC tumor vaccine strategies are being evaluated with modest clinical success.1–3 In vivo DC targeting is an attractive alternative to ex vivo DC vaccines with potential advantages in vaccine efficacy, cost, and availability.2,4,5 Several approaches have been explored to enhance vaccine potency via Ag-presenting cell targeting.6–8 Promising in vivo DC targeting vaccines are being developed such as the use of antibody (Ab) to target DC-specific receptors (e.g., DEC205, DNGR-1, and CD11c) for the delivery of Ag or the use of DC-specific promoter (e.g., CD11c) for the expression of cytokine, leading to the enhancement of vaccine efficacy.2,4,5,9–12

Although considerable effort is now directed toward developing in vivo DC targeting strategies, there is lack of an effective in vivo DC targeting therapeutic antitumor genetic vaccine, which holds a great practical promise to battle tumors because of its safety, low-cost, relatively easy, and fast to make bulk quantities, and ability to activate innate immunity and induce Ag-specific adaptive immunity.6,13 T cell hyporesponsiveness against poorly immunologic self/tumor Ag and tumor-associated Foxp3 CD4 regulatory T cell (Treg)-mediated immune suppression are two major obstacles to an effective tumor vaccine.3,5,14 Indeed, genetic vaccines are rarely effective in self/tumor Ag-based native tumor models even though enormous strategies have been developed to augment their efficacy.15–17 Therefore, identification of novel molecular adjuvants to in vivo “modulate” DC to coordinately render improved CD4 T helper (Th1) and CD8 T cell immunity, and attenuated deleterious Treg effects, is a critical challenge.3,5

Unconventional splicing of transcription factor X-box-binding protein (XBP1) mRNA generates a mature mRNA encoding an active transcription factor (the spliced active form of XBP1: XBP1s).18 XBP1s is constitutively activated in DC and an essential factor required for DC normal development and survival.19,20 Overproduction of XBP1s produces more proinflammatory cytokines after toll-like receptor stimulation in (both murine and human) macrophages and a murine DC line.21–23

In this study, we examined whether in vivo genetic targeting of XBP1s to DC (overexpression of XBP1s by DC: XBP1s/DC) in a genetic vaccine context can potentiate vaccine-induced antitumor efficacy and discerned its pertinent underlying mechanism(s) of action.

Results

Construction and expression of a novel genetic vaccine

We constructed a novel genetic vaccine encoding ubiquitous cytomegalovirus (CMV)-driven transgenic vaccine Aghsp70 [i.e., highly immunogenic model Ag chicken egg ovalbumin (OVA), poorly immunogenic self/tumor Ag murine tyrosinase-related protein 2 (mTRP2) or the extracellular domain of activated oncogene rat Neu (NeuED) fused to human heat shock protein 70: OVAhsp70, mTRP2hsp70, or NeuEDhsp70]12,15,24 and DC-specific CD11c-driven murine XBP1s19 (XBP1s/DC) (Figure 1a). To examine the overproduction of XBP1s and the production of mTRP2hsp70 in DC in vitro, bone marrow (BM)-derived DC (BM-DC) were untreated (endogenous XBP1s expression control and transgenic mTRP2hsp70 negative control) or transfected with XBP1s/DC (mTRP2hsp70 negative control), mTRP2hsp70 (XBP1s constitutive expression control) or XBP1s/DC-mTRP2hsp70 DNA. Seventy two hours later, the production of mTRP2hsp70 and the overproduction of XBP1s in DC were examined by western blot. As shown in Figure 1b, mTRP2hsp70 was effectively expressed in DC transfected with mTRP2hsp70 or XBP1s/DC-mTRP2hsp70 but not XBP1s/DC DNA. As expected, XBP1s was detectable in DC untreated or transfected with mTRP2hsp70 (constitutive expression of XBP1s in DC) and elevated expression of XBP1s was found in DC transfected with XBP1s/DC or XBP1s/DC-mTRP2hsp70 (Figure 1b). To further determine the overexpression of XBP1s and the expression of mTRP2hsp70 in DC in vivo, C57BL/6 (B6) mice were untreated or immunized once using a gene gun (GG) with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA. Seventy two hours later, the constitutive expression or overexpression of XBP1s and the expression of transgenic mTRP2hsp70 (detected by human hsp70) in DC isolated from the draining lymph nodes (DLN) after treatment with genetic vaccines were determined by reverse transcriptase-PCR. As shown in Figure 1c, the constitutive expression of XBP1s was detected in DC from the DLN of mice untreated or immunized with mTRP2hsp70 and the overexpression of XBP1s was verified in DC from the DLN of mice immunized with XBP1s/DC or XBP1s/DC-mTRP2hsp70. The expression of mTRP2hsp70 was detected in DC from the DLN of mice immunized with mTRP2hsp70 or XBP1s/DC-mTRP2hsp70. The data suggest that XBP1s and/or Aghsp70 are effectively expressed in DC engineered by DNA encoding XBP1s and/or Aghsp70.

Figure 1.

Figure 1

Open in a new tab

Vaccine design and protein expression. (a) A novel genetic vaccine encodes ubiquitous cytomegalovirus (CMV)-driven vaccine Aghsp70 and dendritic cells (DC)-specific CD11c-driven XBP1s. (b) Bone marrow-derived DC (BM-DC) were untreated or transfected with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA. Seventy two hours later, mTRP2hsp70, XBP1s, β-actin, and tubulin (the internal-loading control) in DC lysates were detected by western blot (WB). (c) B6 mice (three/group) were untreated or immunized once using a gene gun (GG) with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-TRP2hsp70 DNA. Seventy two hours later, total RNA was purified from DC isolated from the pooled draining lymph nodes (DLN). Mouse XBP1s, human hsp70, and mouse hypoxanthine phosphoribosyltransferase (HPRT) (the internal control) were detected by reverse transcriptase (RT)-PCR. Data (b,c) are representative of two independent experiments with a similar result.

XBP1s/DC improves mTRP2hsp70 genetic vaccine to elicit durable IFN-γ-producing mTRP2-specific Th1 and CD8 T cell responses and CD8-dependent prophylactic antimelanoma B16 immunity

To assess the impacts of XBP1s/DC on mTRP2hsp70 genetic vaccine-induced mTRP2-specific CD4 and CD8 T cell responses, B6 mice were untreated or immunized with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA on days 1, 7, and 14. On days 21 or 60, CD4 and CD8 T cells were isolated from splenocytes of those mice for analysis of mTRP2-specific T cell responses. Purified CD4 or CD8 T cells were restimulated with synergenic BM-DC transduced by lentiviruses expressing mTRP2hsp70 (LV-mTRP2hsp70-DC) or NeuEDhsp70 (LV-NeuEDhsp70-DC, as Aghsp70-specific stimulator control).16 To further confirm mTRP2-specific CD8 T cell responses, in some experiments, splenocytes were restimulated with mTRP2-specific major histocompatibility complex class I peptides (mTRP2180–188) (OVA-specific major histocompatibility complex class I peptides OVA257–264 as Ag-specific control). XBP1s/DC-mTRP2hsp70 was more efficient than mTRP2hsp70 in eliciting long-lasting mTRP2-specific interferon (IFN)-γ-producing CD4 and CD8 T cell responses (Figure 2a and Supplementary Figure S1). Interleukin (IL)-4 and IL-5 were not detectable in the culture supernatants of CD4 T cells stimulated by LV-mTRP2hsp70-DC or LV-NeuEDhsp70-DC (data not shown). To test whether XBP1s/DC-mTRP2hsp70 genetic vaccine-elicited mTRP2-specific T cell responses could lead to antitumor activity, B6 mice were untreated or immunized with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA on days 1, 7, and 14. On day 21, mice were inoculated subcutaneously (s.c.) with exponentially growing native B16 at abdomen. XBP1s/DC did not induce anti-B16 activity (Figure 2b). In consistent with our previous observations,12,15 mice immunized with mTRP2hsp70 slightly but significantly survived longer compared to mice untreated or immunized with XBP1s/DC (Figure 2b). However, XBP1s/DC-mTRP2hsp70 was much more efficient than mTRP2hsp70 in eliciting prophylactic anti-B16 immunity (Figure 2b). Although CD4 depletion at induction phase did not diminish the antitumor immunity, CD8 depletion at effector phase abrogated the observed antitumor immunity (Figure 2c). The data suggest that XBP1s/DC enhances mTRP2hsp70 genetic vaccine to induce long-lasting mTRP2-specific Th1 and CD8 T cell responses and CD8-dependent prophylactic antitumor immunity against native tumors. Although this immunization strategy induced durable mTRP2-specific Th1 and CD8 T cell responses, protective anti-B16 (s.c. model) immunity elicited by this vaccine was CD8- but not CD4-dependent. One possible explanation for this discrepancy is that, in this B16 s.c. model, vaccine-induced Th1 may be not necessary for protective anti-B16 immunity. Treg could be depleted through the administration of anti-CD4 Ab leading to enhanced antitumor immunity. Indeed, CD4 depletion slightly increased CD8-dependent antitumor immunity (Figure 2c).

Figure 2.

Figure 2

Open in a new tab

XBP1s/dendritic cells (DC) improves mTRP2hsp70 genetic vaccine to elicit durable interferon (IFN)-γ-producing mTRP2-specific Th1 and CD8 T cell responses and CD8-dependent prophylactic anti-B16 immunity. B6 mice were untreated or immunized with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA on days 1, 7, and 14. (a) On day 21 or 60, purified CD4 or CD8 T cells were restimulated with LV-mTRP2hsp70-DC or LV-NeuEDhsp70-DC (Aghsp70-specific stimulator control). D3 after restimulation, the concentration of IFN-γ in the culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Data are representative of three (day 21) to four (day 60) independent experiments with a similar result. (b) B6 mice were untreated or immunized as described in (a). On day 21, mice were subcutaneously (s.c.) inoculated with exponentially growing B16. (c) B6 mice were untreated or immunized with XBP1s/DC-mTRP2hsp70 and inoculated B16 as described in (b). Anti-mouse CD4 mAb were injected intraperitonealy (i.p.) on days -3, 2, and 5. Anti-mouse CD8 mAb were injected i.p. on days 20, 22, 25, and 31. Data represent three (b) to four (c) independent experiments. NS, no significant. Animal survival is presented using Kaplan–Meier survival curves.

XBP1s/DC renders mTRP2hsp70 genetic vaccine to elicit therapeutic antitumor immunity against established B16 and glioma GL26

Despite mTRP2hsp70 genetic vaccine failed to elicit therapeutic antitumor immunity against established B16 (Figure 3a), XBP1s/DC rendered this vaccine to inhibit the growth of established B16 (Figure 3a) and to slightly but significantly eradicate established B16 in 2 of 20 tumor-bearing mice (Figure 3b). Effective therapeutic anti-B16 immunity correlated with mTRP2-specific T cell responses (Supplementary Figure S2). In the established GL26 (naturally expressing mTRP2, ref. 25) (~60 mm3) model, mTRP2hsp70 did not effectively inhibit the growth of established GL26 but slightly and significantly prolonged GL26-bearing mice survival (Figure 3c,d). Importantly, XBP1s/DC dramatically improved the therapeutic efficacy of mTRP2hsp70, leading to eradicating established GL26 in 5 of 10 tumor-bearing mice (Figure 3d). The data demonstrate that XBP1s/DC potentiates poorly immunogenic self/tumor Aghsp70 genetic vaccine to elicit therapeutic antitumor immunity against established native tumors.

Figure 3.

Figure 3

Open in a new tab

XBP1s/dendritic cells (DC) renders mTRP2hsp70 genetic vaccine to elicit therapeutic antitumor immunity against established B16 and GL26. (a) B6 mice were inoculated subcutaneously (s.c.) with exponentially growing B16 on day 0. Tumor-bearing mice were randomly allocated to be untreated or vaccinated on days 6 and 13. Data are representative of three independent experiments with a similar result. (b) B16-bearing mice were prepared as described in (a) and were randomly allocated to be untreated or vaccinated on days 6, 13, and 20. Data represent four independent experiments. (c–d) B6 mice were inoculated subcutaneously (s.c.) with exponentially growing GL26 on day 0. Mice bearing the established GL26 (~60 mm3) were randomly allocated to be untreated or vaccinated on days 6, 13, and 20. Data represent two independent experiments. Animal survival is presented using Kaplan–Meier survival curves.

XBP1s/DC potentiates NeuEDhsp70 genetic vaccine to elicit therapeutic CD8-dependent antitumor immunity against established breast tumor 4T1.2-Neu and attenuate tumor-associated Treg suppressive function

During tumor progression, 4T1.2-Neu induces Treg activation.26 XBP1s/DC potentiated NeuEDhsp70 genetic vaccine to elicit CD8-dependent therapeutic antitumor immunity to control tumor growth and to eradicate the established 4T1.2-Neu (Figure 4a,b). The XBP1s/DC-potentiated vaccine did not significantly affect the frequency of Treg but effectively reduced absolute numbers of splenic Treg in tumor-bearing mice (data not shown). Importantly, although Treg from tumor-bearing mice treated by XBP1s/DC-NeuEDhsp70 exhibited suppression function, their suppressive activity was low compared to Treg from tumor-bearing mice untreated or treated by XBP1s/DC or NeuEDhsp70 alone (Figure 4c). The data show that XBP1s/DC-NeuEDhsp70 genetic vaccine induces CD8-dependent therapeutic antitumor immunity against established tumors and attenuates tumor-associated Treg suppressive function.

Figure 4.

Figure 4

Open in a new tab

XBP1s/dendritic cells (DC) potentiates NeuEDhsp70 genetic vaccine to elicit therapeutic CD8-dependent antitumor immunity against established 4T1.2-Neu and attenuate tumor-associated Treg suppressive function. (a–b) BALB/c mice were inoculated subcutaneously (s.c.) with exponentially growing 4T1.2-Neu on day 0. Tumor-bearing mice were randomly allocated to be untreated or vaccinated on days 8, 15, and 22. CD8 depletion was done on days 6, 9, 14, and 21. Data represent two independent experiments. Animal survival is presented using Kaplan–Meier survival curves. (c) Tumor-bearing Foxp3-GFP BALB/c mice were untreated or immunized as described in (a). On day 26, splenic Treg (GFP+) were measured by flow cytometry and sorted. The suppressive activity of sorted Treg was measured. Data are representative of three independent experiments with a similar result.

Overproduction of XBP1s promotes DC survival and enhances mTRP2hsp70-DC maturation and function in vitro

To determine whether overproduction of XBP1s in DC can promote DC survival, BM-DC were untreated or transfected with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA. Viable DC were counted after Trypan blue dye exclusion on days 0, 3, and 9. As expected, overproduction of XBP1s in DC improved DC survival regardless of mTRP2hsp70 (Supplementary Figure S3). To examine whether overproduction of XBP1s in DC can enhance DC maturation, BM-DC were untreated or transfected with vector (backbone control), XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA. Day 3 after transfection, DC and culture supernatants were harvested to determine DC phenotypes and cytokines by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. Overproduction of XBP1s in mTRP2hsp70-DC augmented CD40, CD70, CD86, IL-15, IL-15Rα, and CCR7 expression (Figures 5a and Supplementary Figure S4). Also, DC transfected with XBP1s/DC-mTRP2hsp70 produced significant IL-6, IL-12p40, and tumor necrosis factor (TNF)-α (Figure 5b). To test whether XBP1s/DC can enhance the stimulation of CD8 T cells, vaccine-induced CD8 T cells, isolated from splenocytes of B6 mice immunized with mTRP2hsp70 genetic vaccine, were cocultured with syngenic BM-DC untreated or transfected with XBP1s/DC, mTRP2hsp70 or XBP1s/DC-mTRP2hsp70 DNA. DC transfected with XBP1s/DC-mTRP2hsp70 were more efficient than DC transfected with mTRP2hsp70 in stimulating syngenic vaccine-induced CD8 T cells (Figure 5c). The data suggest that overproduction of XBP1s promotes DC survival and enhances Aghsp70-DC maturation and function in vitro.

Figure 5.

Figure 5

Open in a new tab

Overproduction of XBP1s enhances Aghsp70-dendritic cells (DC) maturation and function in vitro. (a) Bone marrow-derived DC (BM-DC) were untreated or transfected with vector, XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA. Day 3 after DNA transfection, DC were stained by anti-mouse CD11c, CD70, CD86, IL-15Rα, CCR7, or isotype control antibodies (ISO), and analyzed by flow cytometry. One representative of three independent experiments with a similar result is shown. (b) As described in (a), day 3 after transfection, IL-6, IL-12(p40), or TNF-α in culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Medium alone (without DC) is a negative control. Data represent three independent experiments. (c) Vaccine-induced CD8 T cells were obtained from mTRP2hsp70 genetic vaccine-immunized B6 mice. Untreated and DNA-transfected DC were obtained as described in (a). Day 3 after DNA transfection, untreated or DNA-transfected DC were cocultured with vaccine-induced CD8 T cells for 3 days. The proliferation of CD8 T cells was measured. Data represent three independent experiments.

XBP1s/DC elevates functional DEC205+CD8α+DC in the DLN

Unexpectedly, we found that XBP1s/DC elevated DEC205+ CD8α+DC in the DLN (frequency and absolute numbers) regardless of Aghsp70 (Figure 6a,b). Using a well-established OVA-OT-I system, we further show that DEC205+CD8α+DC (but not DEC205+CD8α-DC) were functional in presenting OVA to OT-I (Figure 6c). Despite XBP1s/DC alone elevated DEC205+CD8α+DC (Figure 6a,b), those cells did not exhibit function (due to the lack of OVA) (Figure 6c). Furthermore, the functionality of DEC205+CD8α+DC from the DLN of mice immunized with XBP1s/DC-OVAhsp70 was high compared to DEC205+CD8α+DC from the DLN of mice immunized with OVAhsp70 (Figure 6c). The data suggest that XBP1s/DC improves DEC205+CD8α+DC by increasing their numbers and function.

Figure 6.

Figure 6

Open in a new tab

XBP1s/dendritic cells (DC) elevates functional DEC205+CD8α+DC in the draining lymph nodes (DLN). B6 mice were untreated (nontreatment) or immunized once with vector, XBP1s/DC, Aghsp70 (mTRP2hsp70 or OVAhsp70), or XBP1s/DC-Aghsp70 (XBP1s/DC-mTRP2hsp70 or XBP1s/DC-OVAhsp70) DNA. Seventy two hours later, single-cell suspensions of the DLN were prepared and stained with anti-mouse CD11c-APC, CD8α-FITC and DEC205-PE, and analyzed by flow cytometry. (a) Frequency of DEC205+CD8α+DC in gated CD11c+DC of the DLN: one representative of three independent experiments with a similar result is shown. (b) Absolute numbers of CD11c+DEC205+CD8α+DC in the DLN are shown. (c) Single-cell suspensions of the pooled DLN from B6 mice immunized with vector, XBP1s/DC, OVAhsp70, or XBP1s/DC-OVAhsp70 DNA were obtained and stained as described above. DEC205+CD8α+DC or DEC205+CD8α-DC, sorted from gated CD11c+DC, were cocultured with OT-I cells from Rag2/OT-I mice for 3 days. Interferon (IFN)-γ in the culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Data (b–c) present three independent experiments.

Discussion

We designed a novel tumor genetic vaccine encoding Aghsp70 and XBP1s. Aghsp70 is under a strong ubiquitous CMV because a weak DC-specific CD11c-driven vaccine Ag expression is insufficient to elicit optimal T cell immunity,27 and CMV ensures DC that have taken up the vaccine highly produce the Aghsp70. Although using a strong promoter (e.g., CMV) for the ubiquitous overexpression of XBP1s in host cells including DC may improve vaccine function, restrictedly- (weakly) overproduced XBP1s in vaccine Ag-DC under a weak DC-specific CD11c may avoid, at least significantly reduce, possible adverse effects associated with XBP1s.28 In this vaccine, CMV-driven Aghsp70 and CD11c-driven XBP1s are in a single construct. In the vaccination via a GG, there may be no advantages in using CMV and CD11c promoters in a single construct compared to using two (one for CMV-driven Aghsp70, another for CD11c-driven XBP1s) constructs. However, in the vaccination via intramuscular or intradermal injection, commonly applied in the clinic, there is a potential advantage of in vivo enhancing the chance of simultaneous expression of Aghsp70 and overexpression of XBP1s in the same DC using two promoters in one construct compared to using two separate constructs.

Inducing durable self/tumor Ag-specific T cell responses of high-quality against established tumors has proven very difficult due to two major clinical obstacles: T cell hyporesponsiveness against poorly immunogenic self/tumor Ag and the suppressive microenvironment associated with tumors (i.e., tumor-associated Treg, myeloid derived-suppressor cells, among others).3,5,14,29 The novel targeted codelivery and overproduction of XBP1s with transgenic vaccine Aghsp70 in DC vaccine induced long-lasting mTRP2-specific Th1 and CD8 T cell responses, attenuated tumor-associated Tregs suppressive function, and elicited both prophylactic and therapeutic antitumor immunity in multiple tumor models including melanoma, glioma, and breast tumor, suggesting a novel role for XBP1s in modulating DC function to overcome the aforementioned major obstacles to a successful tumor immunotherapy through synergistic mechanisms.

BM-DC genetically modified to simultaneously express Aghsp70 and overexpress XBP1s demonstrate upregulated expression of CD40, CD70, CD86, IL-15, IL-15Rα, and CCR7, increased secretion of IL-6, IL-12, and TNF-α, and enhanced stimulation of CD8 T cells. This implicates that overproduction of XBP1s comprehensively improves Aghsp70-DC capacities to express costimulatory molecules and chemokine receptor and to produce cytokines, thereby positively influencing DC function for an effective T cell response. Specifically, enhanced CD40/CD86 suggests that XBP1s promotes DC to effectively activate T cells.1 CD70 expression on DC, in particular, immature/semimature DC, which very likely occur in vivo, is important to elicit effective immunity,30 thus, CD70 increased by XBP1s may represent a mechanism of action of XBP1s in improving DC function. XBP1s-enhanced IL15/IL-15Rα signal on DC indicates the generation of effective central memory CD8 T cells,31 which have been reported to mediate potent antitumor impact,32 Increased CCR7 suggests an XBP1s-promoted DC migration into the DLN for the T cell activation.33 Furthermore, improved proinflammatory cytokines (IL-12, IL-6, and TNF-α) suggests that XBP1s-potentiated DC improve T cell immunity and overcome Treg-mediated immune suppression.34–36

XBP1s controls transcription of a number of cell type- or condition-specific targets.20 Overproduction of XBP1s produces more proinflammatory cytokines (e.g., IFN-β, TNF-α, or IL-6) after toll-like receptor stimulation in (both human and mouse) macrophages and a mouse DC line.21–23 Since XBP1s constitutively expressed in DC did not effectively improve their maturation and activation, overproduction of XBP1s in DC may be necessary for direct targeting of costimulatory molecule, cytokine and chemokine receptor genes, leading to the enhanced DC maturation. Indeed, the possible-binding sites of XBP1s on the promoter regions of mouse costimulatory molecule, cytokine and chemokine receptor genes were identified by using the free software MAPPER Search Engine (chip-mapper.org)37 (Supplementary Table S1). These data may help to understand consequence of the signaling initiated by the overexpression of XBP1s in DC. Whether elevated XBP1s in DC is necessary for direct targeting of those genes, in particular, CD70, IL-15Rα, and IL-12p35 is actively investigated. Taken together, the data support the concept that overproduction of XBP1s in DC may promote their capacity to regulate innate and adaptive immune responses. Given that XBP1s overexpression alone did not appear to work in concert with endogenous Ag processing or endogenous hsp70, a necessary interaction with the transgenic Aghsp70 points to a unique underlying mechanism that XBP1s may synergize with Aghsp70 to enhance DC maturation. How this happens is unknown. Although hsp70 is linked to DC activation,38 whether Aghsp70 fusion molecules could initiate similar signaling such as hsp70 alone is uncertain. The mechanism(s) by which XBP1s acts in concert with Aghsp70 to optimize DC function will be examined in the future studies.

Among multiple DC populations in vivo, recent data suggest that CD8α+DC in particular are a preferred DC subset target for cancer vaccines due to their “superior ” capacities to induce Th1 and CD8 T cell responses (e.g., cross-present exogenous Ag, ingest dead/dying cells, production of IL-12p70).39,40 Fms-like tyrosine kinase 3 ligand promotes CD8α+DC expansion.41 However, Fms-like tyrosine kinase 3 ligand also promotes Treg expansion42 that may limit its potentials in developing tumor vaccines. Targeting of Ag to DC via DEC205 has been demonstrated to be effective in enhancing T cell immunity and vaccine potency.9,11 The data show that XBP1s/DC increased the numbers and function of DEC205+CD8α+DC in the DLN, implicating a novel role for XBP1s in improving DEC205+CD8α+DC for potentiating vaccine efficacy. How XBP1s/DC elevates functional DEC205+CD8α+DC is being actively investigated. Whether human XBP1s exerts a similar effect on the recently discovered DNGR-1+BDCA3+ human DC population43—the putative equivalent of mouse CD8α+DC that exhibits potent Ag crosspresenting capability and the most appropriate one to target for cancer vaccines—is worthwhile to be examined. Development of a novel strategy to elevate DNGR-1+BDCA3+ human DC would have a significant impact on development of effective antitumor vaccine approaches in the clinic.

In summary, XBP1s/DC-potentiated tumor vaccine has unique and novel advantages. Specifically, this novel approach addresses both T cell hyporesponsiveness against poorly immunologic self/tumor Ag and tumor-associated Treg-mediated immune suppression. The data implicate novel XBP1s/DC-driven effects we have observed including increased numbers and function of DEC205+CD8α+DC in the DLN and enhanced DC maturation and function. These characteristics are likely relevant to the accentuation of Th1 and CD8 T cell responses, the attenuation of tumor-associated Treg suppressive function, and the potentiation of prophylactic and therapeutic antitumor immunity against native tumors, indicating this vaccine strategy may be potentially translatable into a clinically relevant setting.

Materials and Methods

Mice and cell lines. B6, B6-Rag2/OT-I, BALB/c, BALB/c-Foxp3-GFP (C.Cg-Foxp3tm2Tch/J) mice (female, 6–8 weeks) were purchased from Taconic (Rensselaer, NY) or JAX (Bar Harbor, ME) and housed in specific pathogen-free conditions in the University of Pittsburgh animal facility (Pittsburgh, PA). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals. Murine melanoma B16 (ATCC, Manassas, VA), glioma GL26,25 and breast tumor 4T1.2-Neu24 were maintained in DMEM (IRVINE Scientific, Santa Ana, CA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 2 mmol/l glutamine (Invitrogen, Carlsbad, CA) and 1× antibiotic antimycotic solution (Sigma, St Louis, MO).

Vector constructions. Restricted enzymes, T4 DNA ligase and antarctic phosphatase were purchased from NEB (Ipswich, MA). AccuPrime pfx DNA polymerase and one shot Top10 chemically competent Escherichia coli were purchased from Invitrogen. Primers were synthesized by IDTDNA (Coralville, IA). XBP1s was amplified by PCR using primers (5′-TCAGAATTCGGCGTAGACGT TTCCTGGCTATGG-3′ and 5′-CAGGAATTCAGACAGGCCTATGCTA TCCTCTAG-3′) and plasmid XBP1s RV (a gift from Dr Laurie H. Glimcher at Harvard Medical School) (XBP1: NM_013842).19 PCR-amplified XBP1s was cloned into EcoRI-digested plasmid CMV-OVAhsp70-CD11c12 by blunt-end ligation (resultant vector CMV-OVAhsp70-CD11c-XBP1s). Plasmid CMV-OVAhsp70-CD11c-XBP1s was digested using AscI and self-ligated (resultant vector CMV-CD11c-XBP1s). Plasmid CMV-mTRP2hsp70-CD11c-IL-1512 was digested using AscI (resultant AscI-digested fragment mTRP2hsp70). AscI-digested fragment mTRP2hsp70 was cloned into AscI-digested vector CMV-CD11c12 or CMV-CD11c-XBP1s (resultants vectors CMV-mTRP2hsp70-CD11c and CMV-mTRP2hsp70-CD11c-XBP1s). Inserted genes were confirmed by both enzyme digestion and DNA sequencing. DNA was purified using EndoFree plasmid kits (QIAGEN, Valencia, CA).

mTRP2hsp70 and XBP1s expression in BM-DC. BM cells (1 × 106/ml) taken from naive B6 mice were cultured in RPMI1640 (IRVINE Scientific) supplemented with 10% FBS, 2 mmol/l glutamine, 1× antibiotic antimycotic solution, recombinant mouse granulocyte-macrophage colony-stimulating factor (1,000 U/ml) and IL-4 (1,000 U/ml) (R&D Systems, Minneapolis, MA).44 On day 5, BM-DC were purified using anti-mouse CD11c microbeads (Miltenyi Biotec, Auburn, CA). Purified DC (1 × 106) in 40 µl medium in a 12-well plate were shot with a bullet (3 µg DNA/bullet) using a GG (Bio-Rad, Hercules, CA) at 260 psi pressure.12,45 After transfection, cells were continually cultured in 1-ml DC culture medium. Three days later, DC were harvested, washed, and lysed using CelLytic M Cell Lysis Reagent (Sigma). mTRP2hsp70 and XBP1s in DC lysates were detected by standard western blot using mouse antihuman hsp70 mAb (SPA-820; Stressgene, Farmingdale, NY) and rabbit anti-mouse XBP1 polyclonal Ab (sc-7160; Santa Cruz, Santa Cruz, CA), respectively. Mouse β-actin and tubulin detected by anti-mouse β-actin and tubulin Ab (Sigma) were used as the internal-loading controls. Immunoreactive protein bands were visualized using appropriate horseradish peroxidase-linked secondary antibodies and SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL).46

Reverse transcriptase-PCR. B6 mice (three/group) were untreated or immunized once at the shaved abdominal region using a GG with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-TRP2hsp70 DNA as described previously.12,47 Seventy two hours later, CD11c+ DC were isolated from the pooled DLN using anti-mouse CD11c microbeads. Total cellular RNA was purified using the RNAqueous-4PCR Kit (Ambion, Austin, TX). Reverse transcriptase-PCR was performed using the Transcriptor One-step reverse transcriptase-PCR kit (Roche, Indianapolis, IN) with primers (i.e., human Hsp70: 5′-TTGGC GCGCCATGGCCAAAGCCGCGGCAGTC-3′ and 5′-TTGGCGCGCCC TAATCTACCTCCTCAATGGT-3′; mouse XBP1s: 5′-TCAGAATTCGG CGTAGACGTTTCCTGGCTATGG-3′ and 5′-CAGGAATTCAGACAG GCCTATGCTATCCTCTAG-3′; mouse hypoxanthine phosphoribosyltransferase as the internal control: 5′-GTTGGATACAGGCCAGACTTTG TTG-3′ and 5′-GAAGGGTAGGCTGGCCTATAGGCT-3′) following the program: reverse transcription for 30 minutes at 50 °C and an initial denaturation for 5 minutes at 94 °C, then followed by 35 cycles of 94 °C for 10 seconds, 55 °C for 30 seconds, and 68 °C for 2 minutes. The PCR products were electrophoresed through a 1.0% agarose gel-containing ethidium bromide and visualized under UV light.

BM-DC survival, phenotypes, and cytokines. BM-DC survival was quantified by using Trypan blue dye exclusion on days 0, 3, and 9 after DNA transfection.19 On day 3 after DNA transfection, the concentration of IL-6, IL-12p40, or TNF-α in DC culture supernatants was determined by ELISA (eBioscience, San Diego, CA and Biolegend, San Diego, CA). To examine DC phenotypes, DC were preincubated with anti-mouse CD16/32, and then stained with anti-mouse CD11c-APC (HL3), CD86-PE (GL1), CD70-PE (FR70), CCR7-PE (4B12), CD40-FITC (3/23), IL-15Rα-FITC (FAB551F), or IL-15-Biotinylated (500-P173Bt) (followed by FITC-SA) (isotype control of each Ab was used in control staining) (eBioscience, BD Biosciences (San Diego, CA), PeproTech (Rocky Hill, NJ), and R&D Systems), and analyzed by flow cytometry on a BD LSRII with CellQuest software (BD Biosciences). The flow cytometric data were analyzed using Flowjo software (Tree star, Ashland, OR).

CD8 T cell proliferation. To obtain vaccine-induced CD8 T cells, B6 mice (three/group) were immunized with DNA encoding mTRP2hsp70 on days 1, 7, and 14 as described above. On day 21, CD8 T cells were isolated from splenocytes of DNA-immunized mice using anti-mouse CD8 microbeads (Miltenyi Biotec). On day 3 after DNA transfection, DNA-transfected DC (untreated DC as control) (6 × 104) were cocultured with purified vaccine-induced CD8 T cells (3 × 105) in 0.2 ml RPMI 1640 10% FBS at 37 °C, 5% CO2 for 72 hours. 3H thymidine (1 µCi/well; Du Pont/New England Nuclear, Boston, MA) was added during the last 16–18 hours of culture. 3H thymidine incorporation was measured using a scintillation counter (Packard, Meriden, CT).

mTRP2-specific CD4 and CD8 T cell responses. B6 mice (three/group) were untreated or immunized with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA on days 1, 7, and 14 as described above. On day 21 or 60, CD4 and CD8 T cells were purified from splenocytes of DNA-immunized or untreated mice using anti-mouse CD4 and CD8 microbeads, respectively. LV-transduced DC expressing mTRP2hsp70 (LV-mTRP2hsp70-DC) or NeuEDhsp70 (LV-NeuEDhsp70-DC) were obtained as described previously.16 Purified CD4 or CD8 T cells (8 × 105) were cocultured with syngenic LV-mTRP2hsp70-DC or LV-NeuEDhsp70-DC (1.6 × 105) in 1 ml RPMI 1640 10% FBS at 37 °C, 5% CO2 for 3 days. To further confirm mTRP2-specific CD8 T cell responses, in some experiments, splenocytes were restimulated with mTRP2-specific major histocompatibility complex class I peptides (mTRP2180–188) (2 µg/ml) (OVA-specific major histocompatibility complex class I peptides OVA257–264 as Ag-specific control) (purity >95%, synthesized and high-performance liquid chromatography purified in the core facility of University of Pittsburgh) for 3 days. The concentration of IFN-γ (CD4 and CD8), IL-4, or IL-5 (CD4) in the culture supernatants was determined by ELISA (BD Biosciences).

Treg suppressive activity. BALB/c-Foxp3-GFP mice (three/group) were s.c. inoculated with 4T1.2-Neu (2 × 104) at the 4th mammary fat pad on day 0 as described previously.24,26 Tumor-bearing mice were randomly allocated to be untreated or immunized with XBP1s/DC, NeuEDhsp70, or XBP1s/DC-NeuEDhsp70 DNA on days 8, 15, and 22 as described above. Day 26, splenic Treg (GFP+) were measured by flow cytometry and sorted using a BD FACSAria High Speed Cell Sorter (BD Biosciences). The suppressive activity of Treg was measured as described previously:26 4T1.2-Neu-primed CD4 (2 × 105), 4T1.2-Neu lysate-loaded naive BALB/c splenic DC (2 × 105) and naive BALB/c splenic CD8 (2 × 105) were cocultured in the presence or absence of sorted Treg (2 × 105) in 200 µl RPMI 1640 10% FBS at 37 °C, 5% CO2 for 2 days. IFN-γ in the culture supernatants was measured by ELISA.

DEC205+CD8α+DC in the DLN. B6 mice (3–5/group) were untreated or immunized once with vector (CMV-CD11c backbone control), XBP1s/DC, Aghsp70 (mTRP2hsp70 or OVAhsp70), or XBP1s/DC-Aghsp70 (XBP1s/DC-mTRP2hsp70 or XBP1s/DC-OVAhsp70) DNA as described above. Seventy two hours later, single-cell suspensions of the DLN were prepared and stained with anti-mouse CD11c-APC (N418), CD8α-FITC (53-6.7), and DEC205-PE (NLDC-145) (isotype control of each Ab was used in control staining) (eBioscience, BD Biosciences, and Biolegend), and analyzed by flow cytometry as described above. In some experiments, DEC205+CD8α+DC or DEC205+CD8α-DC (1 × 104), sorted from gated CD11c+DC of single-cell suspensions of pooled DLN from B6 mice immunized with XBP1s/DC, OVAhsp70, or XBP1s/DC-OVAhsp70 DNA, were cocultured with OT-I cells (5 × 104) from Rag2/OT-I mice in 200 µl RPMI 1640 10% FBS at 37 °C, 5% CO2 for 3 days. IFN-γ in the culture supernatants was determined by ELISA.

Antitumor immunity

Prophylactic anti-B16 immunity: B6 mice (3–5/group) were untreated or immunized with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA on days 1, 7, and 14 as described above. On day 21, mice were inoculated s.c. with exponentially growing B16 (5 × 104) at abdomen. To deplete CD4 T cells, anti-mouse CD4 mAb (GK1.5) (200 µg/injection) were intraperitonealy injected on days –3, 2, and 5. To deplete CD8 T cells, anti-mouse CD8 mAb (53–6.7) (200 µg/injection) were intraperitonealy injected on days 20, 22, 25, and 31. T cell depletion was confirmed by flow cytometry and resulted in >95% reduction of relevant cell types (data not shown).

Therapeutic anti-B16 immunity: B6 mice (3–5/group) were inoculated s.c. with exponentially growing B16 (2.5 × 104) at flank on day 0. Tumor-bearing mice were randomly allocated to be untreated or vaccinated with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA on days 6, 13 (and 20) as described above.

Therapeutic anti-GL26 immunity: B6 mice (five/group) were inoculated s.c. with exponentially growing GL26 (1 × 106) at flank on day 0.16 On day 6, mice bore the established GL26 (~60 mm3). Tumor-bearing mice were randomly allocated to be untreated or vaccinated with XBP1s/DC, mTRP2hsp70, or XBP1s/DC-mTRP2hsp70 DNA on days 6, 13, and 20 as described above.

Therapeutic anti-4T1.2-Neu immunity: BALB/c mice (five/group) were s.c. inoculated with 4T1.2-Neu (2 × 104) at the 4th mammary fat pad on day 0.24,26 Tumor-bearing mice were randomly allocated to be untreated or vaccinated with XBP1s/DC, NeuEDhsp70, or XBP1s/DC-NeuEDhsp70 DNA on days 8, 15, and 22 as described above. To deplete CD8 T cells, anti-mouse CD8 mAb (53–6.7) (200 µg/injection) were intraperitonealy injected on days 6, 9, 14, and 21. Tumors were measured using a digital slide calipers (Fisher Scientific, Pittsburgh, PA) in the two perpendicular diameters every 3 days. Mice were dead naturally or sacrificed when tumor reached 10 mm in mean diameter.

Statistics. Data were statistically analyzed using Student's t-test (Graph Pad Prism version 5). Data from animal survival experiments were statistically analyzed using Log rank test (Graph Pad Prism version 5). P < 0.05 is considered to be statistically significant.

Acknowledgments

We are indebted to L.H. Glimcher (Harvard Medical School) for providing plasmid XBP1s RV and A.-H. Lee (Harvard School of Public Health) for help in designing PCR primers. We also are indebted to R.M. Steinman (The Rockefeller University) and T.C. Wu (John Hopkins Medical Institutions) for their encouragements. This work was supported by NIH grant R01CA108813, R01CA108813-04S2 (to Z.Y.), P50 CA121973 and R01 AI076060 (to L.D.F.). S.T., Z.L., and Z.Y. designed research; S.T., Z.L., and C.D. performed research; S.T., Z.L., L.D.F., and Z.Y. analyzed data; and Z.Y. wrote the paper. The authors declared no conflict of interest.

Supplementary Material

Figure S1

XBP1s/DC improves mTRP2hsp70 genetic vaccine to elicit IFN-γ-producing mTRP2-specific CD8 T cell responses.

Click here for additional data file. (38.6KB, pdf)
Figure S2

Effective therapeutic anti-B16 immunity correlates with mTRP2-specific T cell responses.

Click here for additional data file. (47.4KB, pdf)
Figure S3

Overproduction of XBP1s promotes DC survival in vitro.

Click here for additional data file. (40.9KB, pdf)
Figure S4

Overproduction of XBP1s enhances Aghsp70-DC maturation in vitro.

Click here for additional data file. (57.6KB, pdf)
Table S1

Possible-binding sites of XBP1 on promoters of costimulatory molecule, cytokine, and chemokine receptor genes.

Click here for additional data file. (149.5KB, doc)

REFERENCES

  1. Banchereau J., and, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245–252. doi: 10.1038/32588. [DOI] [PubMed] [Google Scholar]
  2. Melief CJ. Cancer immunotherapy by dendritic cells. Immunity. 2008;29:372–383. doi: 10.1016/j.immuni.2008.08.004. [DOI] [PubMed] [Google Scholar]
  3. Klebanoff CA, Acquavella N, Yu Z., and, Restifo NP. Therapeutic cancer vaccines: are we there yet. Immunol Rev. 2011;239:27–44. doi: 10.1111/j.1600-065X.2010.00979.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Steinman RM. Dendritic cells in vivo: a key target for a new vaccine science. Immunity. 2008;29:319–324. doi: 10.1016/j.immuni.2008.08.001. [DOI] [PubMed] [Google Scholar]
  5. Palucka K, Ueno H., and, Banchereau J. Recent developments in cancer vaccines. J Immunol. 2011;186:1325–1331. doi: 10.4049/jimmunol.0902539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Tsen SW, Paik AH, Hung CF., and, Wu TC. Enhancing DNA vaccine potency by modifying the properties of antigen-presenting cells. Expert Rev Vaccines. 2007;6:227–239. doi: 10.1586/14760584.6.2.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kutzler MA., and, Weiner DB. Developing DNA vaccines that call to dendritic cells. J Clin Invest. 2004;114:1241–1244. doi: 10.1172/JCI23467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. You Z, Huang X, Hester J, Toh HC., and, Chen SY. Targeting dendritic cells to enhance DNA vaccine potency. Cancer Res. 2001;61:3704–3711. [PubMed] [Google Scholar]
  9. Nchinda G, Kuroiwa J, Oks M, Trumpfheller C, Park CG, Huang Y.et al. (2008The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells J Clin Invest 1181427–1436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Sancho D, Mourão-Sá D, Joffre OP, Schulz O, Rogers NC, Pennington DJ.et al. (2008Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin J Clin Invest 1182098–2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Caminschi I, Lahoud MH., and, Shortman K. Enhancing immune responses by targeting antigen to DC. Eur J Immunol. 2009;39:931–938. doi: 10.1002/eji.200839035. [DOI] [PubMed] [Google Scholar]
  12. Tian S, Liu Z, Donahue C, Noh HS, Falo LD., Jr, and, You Z. Transcriptional IL-15-directed in vivo DC targeting DNA vaccine. Gene Ther. 2009;16:1260–1270. doi: 10.1038/gt.2009.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Rice J, Ottensmeier CH., and, Stevenson FK. DNA vaccines: precision tools for activating effective immunity against cancer. Nat Rev Cancer. 2008;8:108–120. doi: 10.1038/nrc2326. [DOI] [PubMed] [Google Scholar]
  14. Zou W. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol. 2006;6:295–307. doi: 10.1038/nri1806. [DOI] [PubMed] [Google Scholar]
  15. Kim JH, Chen J, Majumder N, Lin H, Falo LD., Jr, and, You Z. 'Survival gene‘ Bcl-xl potentiates DNA-raised antitumor immunity. Gene Ther. 2005;12:1517–1525. doi: 10.1038/sj.gt.3302584. [DOI] [PubMed] [Google Scholar]
  16. Kim JH, Majumder N, Lin H, Watkins S, Falo LD., Jr, and, You Z. Induction of therapeutic antitumor immunity by in vivo administration of a lentiviral vaccine. Hum Gene Ther. 2005;16:1255–1266. doi: 10.1089/hum.2005.16.1255. [DOI] [PubMed] [Google Scholar]
  17. Engelhorn ME, Guevara-Patiño JA, Merghoub T, Liu C, Ferrone CR, Rizzuto GA.et al. (2008Mechanisms of immunization against cancer using chimeric antigens Mol Ther 16773–781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Yoshida H, Matsui T, Yamamoto A, Okada T., and, Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell. 2001;107:881–891. doi: 10.1016/s0092-8674(01)00611-0. [DOI] [PubMed] [Google Scholar]
  19. Iwakoshi NN, Pypaert M., and, Glimcher LH. The transcription factor XBP-1 is essential for the development and survival of dendritic cells. J Exp Med. 2007;204:2267–2275. doi: 10.1084/jem.20070525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Todd DJ, Lee AH., and, Glimcher LH. The endoplasmic reticulum stress response in immunity and autoimmunity. Nat Rev Immunol. 2008;8:663–674. doi: 10.1038/nri2359. [DOI] [PubMed] [Google Scholar]
  21. Smith JA, Turner MJ, DeLay ML, Klenk EI, Sowders DP., and, Colbert RA. Endoplasmic reticulum stress and the unfolded protein response are linked to synergistic IFN-β induction via X-box binding protein 1. Eur J Immunol. 2008;38:1194–1203. doi: 10.1002/eji.200737882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Martinon F, Chen X, Lee AH., and, Glimcher LH. TLR activation of the transcription factor XBP1 regulates innate immune responses in macrophages. Nat Immunol. 2010;11:411–418. doi: 10.1038/ni.1857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hu F, Yu X, Wang H, Zuo D, Guo C, Yi H.et al. (2011ER stress and its regulator X-box-binding protein-1 enhance polyIC-induced innate immune response in dendritic cells Eur J Immunol 411086–1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kim JH, Majumder N, Lin H, Chen J, Falo LD., Jr, and, You Z. Enhanced immunity by NeuEDhsp70 DNA vaccine Is needed to combat an aggressive spontaneous metastatic breast cancer. Mol Ther. 2005;11:941–949. doi: 10.1016/j.ymthe.2005.01.003. [DOI] [PubMed] [Google Scholar]
  25. Prins RM, Odesa SK., and, Liau LM. Immunotherapeutic targeting of shared melanoma-associated antigens in a murine glioma model. Cancer Res. 2003;63:8487–8491. [PubMed] [Google Scholar]
  26. Liu Z, Kim JH, Falo LD., Jr, and, You Z. Tumor regulatory T cells potently abrogate antitumor immunity. J Immunol. 2009;182:6160–6167. doi: 10.4049/jimmunol.0802664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lauterbach H, Gruber A, Ried C, Cheminay C., and, Brocker T. Insufficient APC capacities of dendritic cells in gene gun-mediated DNA vaccination. J Immunol. 2006;176:4600–4607. doi: 10.4049/jimmunol.176.8.4600. [DOI] [PubMed] [Google Scholar]
  28. Carrasco DR, Sukhdeo K, Protopopova M, Sinha R, Enos M, Carrasco DE.et al. (2007The differentiation and stress response factor XBP-1 drives multiple myeloma pathogenesis Cancer Cell 11349–360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Gabrilovich DI., and, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol. 2009;9:162–174. doi: 10.1038/nri2506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Keller AM, Schildknecht A, Xiao Y, van den Broek M., and, Borst J. Expression of costimulatory ligand CD70 on steady-state dendritic cells breaks CD8+ T cell tolerance and permits effective immunity. Immunity. 2008;29:934–946. doi: 10.1016/j.immuni.2008.10.009. [DOI] [PubMed] [Google Scholar]
  31. Mortier E, Advincula R, Kim L, Chmura S, Barrera J, Reizis B.et al. (2009Macrophage- and dendritic-cell-derived interleukin-15 receptor α supports homeostasis of distinct CD8+ T cell subsets Immunity 31811–822. [DOI] [PubMed] [Google Scholar]
  32. Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K, Cardones AR, Finkelstein SE.et al. (2005Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells Proc Natl Acad Sci USA 1029571–9576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ohl L, Mohaupt M, Czeloth N, Hintzen G, Kiafard Z, Zwirner J.et al. (2004CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions Immunity 21279–288. [DOI] [PubMed] [Google Scholar]
  34. Colombo MP., and, Trinchieri G. Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev. 2002;13:155–168. doi: 10.1016/s1359-6101(01)00032-6. [DOI] [PubMed] [Google Scholar]
  35. Pasare C., and, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science. 2003;299:1033–1036. doi: 10.1126/science.1078231. [DOI] [PubMed] [Google Scholar]
  36. Song XT, Evel-Kabler K, Shen L, Rollins L, Huang XF., and, Chen SY. A20 is an antigen presentation attenuator, and its inhibition overcomes regulatory T cell-mediated suppression. Nat Med. 2008;14:258–265. doi: 10.1038/nm1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Marinescu VD, Kohane IS., and, Riva A. The MAPPER database: a multi-genome catalog of putative transcription factor binding sites. Nucleic Acids Res. 2005;33 Database issue:D91–D97. doi: 10.1093/nar/gki103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Millar DG, Garza KM, Odermatt B, Elford AR, Ono N, Li Z.et al. (2003Hsp70 promotes antigen-presenting cell function and converts T-cell tolerance to autoimmunity in vivo Nat Med 91469–1476. [DOI] [PubMed] [Google Scholar]
  39. Hildner K, Edelson BT, Purtha WE, Diamond M, Matsushita H, Kohyama M.et al. (2008Batf3 deficiency reveals a critical role for CD8α+ dendritic cells in cytotoxic T cell immunity Science 3221097–1100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shortman K., and, Heath WR. The CD8+ dendritic cell subset. Immunol Rev. 2010;234:18–31. doi: 10.1111/j.0105-2896.2009.00870.x. [DOI] [PubMed] [Google Scholar]
  41. Maraskovsky E, Brasel K, Teepe M, Roux ER, Lyman SD, Shortman K.et al. (1996Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified J Exp Med 1841953–1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Swee LK, Bosco N, Malissen B, Ceredig R., and, Rolink A. Expansion of peripheral naturally occurring T regulatory cells by Fms-like tyrosine kinase 3 ligand treatment. Blood. 2009;113:6277–6287. doi: 10.1182/blood-2008-06-161026. [DOI] [PubMed] [Google Scholar]
  43. Villadangos JA., and, Shortman K. Found in translation: the human equivalent of mouse CD8+ dendritic cells. J Exp Med. 2010;207:1131–1134. doi: 10.1084/jem.20100985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. You Z, Huang XF, Hester J, Rollins L, Rooney C., and, Chen SY. Induction of vigorous helper and cytotoxic T cell as well as B cell responses by dendritic cells expressing a modified antigen targeting receptor-mediated internalization pathway. J Immunol. 2000;165:4581–4591. doi: 10.4049/jimmunol.165.8.4581. [DOI] [PubMed] [Google Scholar]
  45. Larregina AT, Morelli AE, Tkacheva O, Erdos G, Donahue C, Watkins SC.et al. (2004Highly efficient expression of transgenic proteins by naked DNA-transfected dendritic cells through terminal differentiation Blood 103811–819. [DOI] [PubMed] [Google Scholar]
  46. Liu Z, Falo LD., Jr, and, You Z. Knockdown of HMGB1 in tumor cells attenuates their ability to induce regulatory T cells and uncovers naturally acquired CD8 T cell-dependent antitumor immunity. J Immunol. 2011;187:118–125. doi: 10.4049/jimmunol.1003378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Condon C, Watkins SC, Celluzzi CM, Thompson K., and, Falo LD., Jr DNA-based immunization by in vivo transfection of dendritic cells. Nat Med. 1996;2:1122–1128. doi: 10.1038/nm1096-1122. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

XBP1s/DC improves mTRP2hsp70 genetic vaccine to elicit IFN-γ-producing mTRP2-specific CD8 T cell responses.

Click here for additional data file. (38.6KB, pdf)
Figure S2

Effective therapeutic anti-B16 immunity correlates with mTRP2-specific T cell responses.

Click here for additional data file. (47.4KB, pdf)
Figure S3

Overproduction of XBP1s promotes DC survival in vitro.

Click here for additional data file. (40.9KB, pdf)
Figure S4

Overproduction of XBP1s enhances Aghsp70-DC maturation in vitro.

Click here for additional data file. (57.6KB, pdf)
Table S1

Possible-binding sites of XBP1 on promoters of costimulatory molecule, cytokine, and chemokine receptor genes.

Click here for additional data file. (149.5KB, doc)

Articles from Molecular Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

RESOURCES