The Role of IL-12 Signaling in Enhanced Anti-HIV Immunity

<div><p>(A and B) In vivo injection with IL-12 preferentially enhanced gp120-specific CTL and Th responses induced by SOCS1-silenced DCs. C57BL/6 mice were immunized with 1 × 10⁶ of HIV gp120-pulsed (50 μg/ml), transduced DCs derived from BM of WT mice or IL-12 receptor KO mice with ex vivo TNFα maturation (50 ng/ml). On days 1, 3, and 5 after DC immunization, murine IL-12 (1 μg/mouse, Peprotech) was administered intraperitoneally. CD8⁺ T cells (A) or CD4⁺ T cells (B) isolated 2 wk later from the pooled splenocytes of immunized mice (2–3 each group) were subjected to IFN-γ ELISPOT assays. An irrelevant protein, OVA, was used as a negative control. Representative data from two independent experiments are presented. *<i>P</i> < 0.01, LV-SOCS1-siRNA-DC versus LV-SOCS1-siRNA-DC + IL-12, or IL12R KO LV-SOCS1-siRNA-DC + IL-12 versus LV-SOCS1-siRNA-DC + IL-12.</p> <p>(C and D) gp120-specific CTL and Th responses induced by SOCS1-silenced DCs or Ad-IL-12-DCs. BM-derived DCs from WT mice were transfected with LV-SOCS1-siRNA (MOI of 5) or Ad-IL-12 with various MOIs of 10–1,000 or cotransfected with LV-SOCS1-siRNA (MOI of 5) and Ad-IL-12 (MOI of 10) for 4 h. DCs derived from BM of IL-12 receptor KO mice were cotransfected with LV-SOCS1-siRNA (MOI of 5) and Ad-IL-12 (MOI of 10) for 4 h. Groups of C57BL/6 mice were immunized with 1 × 10⁶ of gp120-pulsed (50 μg/ml), transfected DCs with ex vivo TNFα maturation. CD8⁺ T-cells (C) or CD4⁺ T cells (D) isolated 2 wk later from the pooled splenocytes of immunized mice (2–3 each group) were subjected to IFN-γ ELISPOT assays. An irrelevant protein, OVA, was used as a negative control. Representative data from two independent experiments are presented. <i>P</i> < 0.01, Ad-IL-12/SOCS1-siRNA-DC versus IL-12-DCs, or Ad-IL-12/SOCS1-siRNA-DC versus IL12R KO Ad-IL-12/SOCS1-siRNA-DC.</p></div

Enhanced gp120-Specific Antibody and T Cell Responses Induced by SOCS1-Silenced DCs

<p>Groups of C57BL/6 mice were immunized with gp120 (SF162) protein-pulsed, transduced BM-derived DCs (1 × 10⁶ cells/mouse) twice at a weekly interval, followed by PolyI:C or R837 stimulation (30 μg/mouse) in vivo three times on days 1, 3, and 5 after each DC immunization, and sera and splenocytes were collected from each group of mice 14 d later. HIV gp120-specific IgG subclass titers (A) from the pooled sera of each group (4–6 mice/group) were quantified by capture ELISA. CD8⁺ T cells (B) and CD4⁺ T cells (C) isolated from pooled splenocytes were used for IFN-γ ELISPOT assays stimulated with gp120 proteins. Intracellular IFN-γ staining of CD8⁺ T cells from the pooled splenocytes were also performed (D). Representative data from one of three experiments are presented. NS, no stimulation. *<i>P</i> < 0.01, LV-SOCS1-siRNA-DCs versus LV-GFP-siRNA-DCs.</p

Resistance of SOCS1-Silenced DCs to HIV gp120-Mediated Suppression

<div><p>(A) Effects of gp120 on cytokine production by DCs. BM-DCs were transfected with SOCS1-siRNA or a SOCS1-siRNA mutant oligonucleotide as described previously [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030011#pmed-0030011-b024" target="_blank">24</a>], and then cultured with or without SF162 gp120 (20 μg/ml) or LPS (100 ng/ml), and cytokine levels were determined at the different times of cultures, as indicated.</p> <p>(B–E) Effects of gp120 on DC antigen presentation in vivo. Transfected BM-DCs were pulsed with OVA, incubated with or without gp120 for 2 d, and then stimulated with LPS (100 ng/ml) ex vivo overnight. Mice were then immunized with the transduced DCs twice, following three in vivo LPS stimulations. OVA-specific antibody IgG (B) and IgG1 (C) titers and frequencies of IFN-γ-producing OVA-specific CD8⁺ T cells (D) and CD4⁺ T cells (E) were examined 2 wk after the second DC immunization. Data are representative of two repeats.</p></div

Enhanced Production of Both Th1- and Th2-Polarizing Cytokines by SOCS1-Silenced DCs and Activated CD4⁺ Th

<div><p>(A) Enhanced production of both Th1- and Th2-polarizing cytokines by SOCS1-silenced DCs. BM-DCs transfected with SOCS1 siRNA or control [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030011#pmed-0030011-b024" target="_blank">24</a>] were stimulated with LPS (100 ng/ml). Concentrations of various cytokines in the culture media were analyzed by ELISA 24 h after stimulation. NS, no stimulation. *<i>P</i> < 0.01, LV-SOCS1-siRNA-DCs versus LV-GFP-siRNA-DCs.</p> <p>(B) IL-12 production by transduced DCs. BM cells were cultured with mGM-CSF (20 μg/ml only or mGM-CSF and mIL-4 (20 μg/ml) [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030011#pmed-0030011-b024" target="_blank">24</a>] for 6 d and then transduced with LV-SOCS1-siRNA or LV-GFP-siRNA. The transduced DCs (5 × 10⁵/ml) were then stimulated with LPS (100 ng/ml) and plate-coated anti-CD40 mAb (5 μg/ml, BD Bioscience) in the presence or absence of IL-4. Concentrations of IL-12 (p70) in the culture media were analyzed by ELISA 24 h after stimulation and are presented from one of three independent experiments. *<i>P</i> < 0.01, LV-SOCS1-siRNA (GM-CSF + IL-4) versus LV-GFP-siRNA (GM-CSF + IL-4).</p> <p>(C–E) CD4⁺ Th responses induced by LV-SOCS1-siRNA-DCs. CD4⁺ T cells were isolated from pooled splenocytes of different groups of mice and subjected to the following assays. Numbers of IFN-γ-producing CD4⁺ T cell precursors were determined with the ELISPOT assay (C). ³H-thymidine incorporation rates of the isolated CD4⁺ T cells were determined on the fourth day of restimulation with gp120-pulsed DCs (D). Cytokine levels in the culture medium of isolated CD4⁺ cells stimulated with gp120-pulsed DCs for 48 h were determined by ELISA (E). The mean results (+ standard error) from one of three experiments are presented. *<i>P</i> < 0.01, LV-SOCS1-siRNA-DC versus LV-GFP-siRNA-DC mice.</p></div

Enhanced HIV-Specific B Cell Responses

<div><p>(A) Enhanced production of BAFF and APRIL by SOCS1-silenced DCs. The transduced BM-DCs were stimulated with LPS (100 ng/ml) for 24 h. Relative expression levels of BAFF and APRIL mRNA were then determined by real-time quantitative PCR as described in Methods, and normalized to mock-transfected DCs after LPS stimulation using the Comparative Ct method [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030011#pmed-0030011-b027" target="_blank">27</a>]. Representative data from two independent experiments are presented. *<i>P</i> < 0.01, LV-SOCS1-siRNA-DC versus LV-GFP-siRNA-DCs.</p> <p>(B–D) Enhanced activation of gp120-specific B cells by SOCS1-silenced DCs. Frequencies of anti-gp120 antibody-producing cells in different groups of mice were determined and reported as the number of cells secreting gp120-specific IgG per 5 × 10⁵ B cells (B). The proliferation rates (C) and cytokine production (D) of B cells (5 × 10⁴/well) isolated from the spleens of different groups of mice after stimulation with anti-CD40 (5 μg/ml), IL-4 (20 ng/ml), or costimulation with anti-CD40 and IL-4 for 48 h were determined, and results from one of three independent experiments are presented. <i>P</i> < 0.01, LV-SOCS1-siRNA-DC mice versus LV-GFP-siRNA-DC mice under various in vitro stimulation conditions.</p></div

Comparison of gp120-Specific Antibody and T Cell Responses Induced by Protein Immunization and SOCS1-Silenced DCs

<p>Groups of C57BL/6 mice were immunized with gp120 protein (20 μg/ml)-pulsed, transduced BM-derived DCs (1 × 10⁶ cells/mouse) or the same amount of gp120 protein formulated in IFA (20 μg/mouse) twice at a weekly interval. All of the mice were injected with PolyI:C or R837 (30 μg/mouse) in vivo three times on days 1, 3, and 5 after each immunization, and sera and splenocytes were collected from each group of mice 14 d later. HIV gp120-specific IgG subclass titers (A) from the pooled sera of each group (4–6 mice/group) were quantified by capture ELISA. CD8⁺ T cells (B) and CD4⁺ T cells (C) isolated from pooled splenocytes were used for IFN-γ ELISPOT assays stimulated with gp120 proteins. Representative data from one of three experiments are presented. *<i>P</i> < 0.01, gp120 protein + IFA versus gp120-pulsed LV-SOCS1-siRNA-DCs.</p

Long-Term gp120-Specific Antibody and CTL Responses Induced by SOCS1-Silenced DCs

<p>IgG subclass titers (A) from pooled sera of different groups of mice and frequencies of IFN-γ-positive T cells of CD8⁺ T cells (B) and CD4⁺ T cells (D) isolated from pooled splenocytes (two mice per group) were determined at 6 mo after DC immunization and on day 7 after booster immunization with recombinant gp120 emulsified in IFA (20 μg protein/mouse). Intracellular IFN-γ and surface CD44 costaining of gated CD8⁺ T cells from splenocytes at 6 mo after immunization are shown (C). The data are representative of two experiments.</p

Enhancement of HIV DNA Vaccine by SOCS1-siRNA DNA

<p>Groups of C57BL/6 mice were immunized with gp140CF DNA [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030011#pmed-0030011-b074" target="_blank">74</a>] only or a mixture of gp140CF DNA and pSuper-SOCS1-siRNA expressor DNA [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030011#pmed-0030011-b024" target="_blank">24</a>] weekly for 3 wk, followed by PolyI:C or R837 stimulation (30 μg/mouse) in vivo three times on days 1, 3, and 5 after each DNA immunization. HIV gp120-specific IgG subclass titers (A), IFN-γ spot numbers of CD8⁺ T cells (B) and CD4⁺ T cells (C) from pooled splenocytes of different groups of mice (4–6 mice per group) 1 wk after the last immunization are shown from one of three independent experiments. Intracellular IFN-γ staining of CD8⁺ T cells from the pooled splenocytes was also performed (D). *<i>P</i> < 0.01, gp140CF and GFP siRNA versus gp140CF and SOCS1 siRNA coimmunization.</p