A20 controls maturation and cytokine production of Mф.

<p><b>A</b>. Expression of costimulatory molecules and MHC class II molecule on the adenoviral-transduced BMMф in response to stimulation of LPS. <b>B</b>. Production of inflammatory cytokines by the adenoviral-transduced BMMфs, as tested by ELISA. <b>C</b>. NO production by adenoviral-transduced BMMфs, as tested by Griess assay. Experiments were repeated three times with similar results. *p<0.05, **p<0.01 Ad-shA20- vs. Ad-con-transduced Mф.</p

A20-silenced Mф immunization enhances NK cell-, CD8⁺ T cell- and CD4⁺ T cell-mediated cytotoxicity.

<p>Splenocytes pooled from 2–3 immunized mice were cultured overnight for NK-mediated cytotoxicity assay or 5–6 days in the presence of OT-I or OT-II peptide for T cells-mediated cytotoxicity assay. The splenocytes cultured with OT-II peptide were selected using anti-CD4 beads prior to cytotoxicity assay. Cytotoxic activities were analyzed by LDH release assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048930#s4" target="_blank">Material and Methods</a>. Experiments were repeated three times with similar results. *p<0.05, Ad-shA20-Mф immunization vs. Ad-con-Mф immunization for specific killing.</p

A20-silenced Mф elicits a cytotoxic CD4⁺ T cell response via activation of IFN-γ signaling and by an MHC-class-II-restricted mechanism.

<p>A. Adenoviral-transduced BMMфs were used to immunize IFNGR^−/− mice or the wildtype littermates (2–3 mice/group) twice. The inguinal LNs were harvested for analyzing expression of granzyme B in CD4⁺ or CD8⁺ T cells by ICS. p<0.01 Ad-shA20-IFNGR KO mice vs. Ad-ShA20 WT mice. B. Adenoviral-transduced BMMфs were used to immunize Stat1^−/− mice or the wild-type littermates twice (2–3 mice/group). The LNs were harvested for analyzing expression of granzyme B in CD4⁺ (p<0.05, Ad-shA20-Stat1 KO mice vs. Ad-shA20-WT mice) or CD8⁺ T cells by ICS. C. BMMфs were prepared from MHCII^−/− mice or the wild-type littermates. The adenoviral-transduced BMMфs were used to immunize wild-type mice (2–3 mice/group) twice. The LNs were harvested for analyzing expression of granzyme B in CD4⁺ (p<0.01, Ad-shA20-MHC-II KO Mф immunization vs. Ad-shA20-WT Mф immunization) or CD8⁺ T cells by ICS. Experiments were repeated with similar results.</p

IFN-γ enhances MФ to prime cytotoxic T cells response <i>in vitro.</i>

<p>BMMфs were transduced with Ad-con and cocultured with CD4⁺ OT-II (<b>A</b>) or CD8⁺ OT-I (<b>B</b>) T cells in the presence of the different doses of IL-6, IL-12 or IFN-γ (2.5 ug/ml or 10 ug/ml ) for 3–5 days. A20-silenced MФ priming OT-II or OT-I T cells was used as positive control. Expression of granzyme B in T cells was assessed by ICS assay. The data is a representative of three independent experiments. p<0.01, OT-II/con-Mф+IFN-γ(10 ug/ml) vs. OT-II/con-Mф or OT-I/con-Mф+IFN-γ (10 ug/ml) vs. OT-I/con-Mф.</p

A20-silenced Mф enhances expression of granzyme B in CD4⁺ T cells, CD8⁺T cells or NK cells.

<p><b>A,</b> adenoviral-transduced BMMфs were cocultured with freshly isolated OT-I (<b>uppe</b>r) or OT-II cells (<b>lower</b>) at a raito of 1∶10. 3–5 days later, the cocultured T cells were harvested for analyzing expression of granzyme B by ICS. The data is shown as a representative of 3 independent experiments. (p<0.05, OT-I/shA20-Mф vs. OT-I/con- Mф; p<0.01, OT-II/shA20-Mф vs. OT-II/con-Mф). <b>B,</b> C57BL/6 mice (5–6 mice/group) were immunized (<i>i.p</i>) twice with different adenoviral-transduced Mфs or PBS. Lymphocytes were isolated from the inguinal LNs to analyze expression of granzyme B in NK cells, CD8<b>⁺</b> or CD4<b>⁺</b> T cells by ICS. <b>C.</b> C57BL/6 mice were immunized (<i>i.p</i>) twice with OT-II-pulsed, different adenoviral-transduced BMMфs or PBS. Splenocytes were harvested and in vitro restimulated with OT-II peptide for 48 hrs. CD4⁺ T cells were isolated for analysis of granzyme B expression by qPCR. The data is shown as a representation of three independent experiments. (* p<0.01, shA20- Mф-mice vs. con- Mф-mice).</p

A20-silenced Mф immunization induces enhanced immune protection.

<p><b>A & B.</b> C57BL/6 mice (5–6 mice/group) were immunized twice. The mice were s.c. injected with 5×10⁵ EG-7 (<b>A</b>) or M05 (<b>B</b>). Tumor growth was monitored on the indicated days. * p<0.05, Ad-shA20-Mф immunization vs. Ad-con-Mф immunization. <b>C</b>. CD4^−/− C57BL/6 or the wildtype littermates (5–6 mice/group) were immunized with OT-II-peptide-pulsed, Ad-shA20-transduced BMMфs twice followed by s.c. injection of 5×10⁵ M05 tumor cells. Tumor occurrence and growth were monitored on the indicated days. **p<0.01, wild-type mice vs. CD4^−/− mice. <b>D</b>. Transferred OT-II-specific immune pretection. In vitro primed OT-II T cells (5×10⁶) were transplanted into naïve RAG^−/−C57BL/6 mice (5 mice/group) by retro-orbital injection following s.c injection of OVA-expressed B6SJ1003 tumor cells (6×10⁵). The transplantation of OT-II T cells was repeated one week later. One group of mice were transplanted with CMA-treated, Ad-shA20-transduced Mф-primed OT-II T cells. Tumor growth was monitored on the indicated days. *p<0.05, Ad-shA20-Mф-primed OT-II T cell transfer vs. Ad-con-Mф-primed OT-II T cell transfer, or Ad-shA20-Mф-primed OT-II T cell transfer vs. Ad-shA20-Mф-primed OT-II T cell+ CMT transfer. All the experiments were repeated with similar results.</p

Neutralization of IFN-γ reduces A20-silenced MФ to prime cytotoxic T cell response <i>in vitro.</i>

<p>BMMфs were transduced with Ad-shA20 and cocultured with CD4⁺ OT-II (<b>A</b>) or CD8⁺ OT-I (<b>B</b>) T cells in the presence of the different doses of anti-IL-6, anti-IL-12 or anti-IFN-γ (2.5 ug/ml, 10 ug/ml, or 20 ug/ml ) for 3–5 days. Expression of granzyme B in T cells was assessed by ICS assay. The data is a representative of three independent experiments. p<0.01, OT-II/AdshA20-Mф vs. OT-II/AdshA20-Mф+anti-IFN-γ(20 ug/ml).</p

IFN-γ impacts MФ to trigger cytotoxic T cell responses in immunized mice.

<p>C57BL/6 mice (2–3 mice per group) were immunized twice with 1, PBS plus IgG; 2, PBS plus IFN-γ; 3, Ad-con-Mф; 4, Ad-con-Mф plus IFN-γ; 5, Ad-shA20-Mф plus IgG; or 6, Ad-shA20-Mф plus anti-IFN-γ. Two weeks after the 2^nd immunization, inguinal lymph nodes were harvested to analyze expression of granzyme B in CD4⁺ T cells (<b>A</b>) (p<0.05, shA20-Mф+ anti-IFN-γ immunization vs. shA20-Mф+IgG immunization; p<0.01, con-Mф+ IFN-γ immunization vs. con-Mф immunization) or CD8⁺ T cells (<b>B</b>) (p<0.01, shA20-Mф+ anti-IFN-γ immunization vs. shA20-Mф+IgG immunization; p<0.05, con-Mф+ IFN-γ immunization vs. con-Mф immunization) by ICS assay.</p

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