1. Genetic Targeting of the Active Transcription Factor XBP1s to Dendritic Cells Potentiates Vaccine-induced Prophylactic and Therapeutic Antitumor Immunity 
Shenghe Tian, Zuqiang Liu, Cara Donahue, Louis D Falo, Zhaoyang You 
Molecular Therapy 
Volume 20, Issue 2, Pages (February 2012)
DOI: /mt
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 5
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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 6
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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions