Decreased HL expression in Ad-LIGHT infected mice is independent of T cells and IL-1β.

<p>Mice were injected with PBS or adenoviral vectors (1.25×10⁹ pfu/mouse) and sacrificed on the 7^th day. (A) Liver HL mRNA expression in LIGHT adenovirus (Ad-L), human apoA-I adenovirus (Ad-AI) or PBS (P) injected LDLR^−/− mice was analyzed by real time PCR. (B) Morphology of liver from PBS and adenoviral infected mice (H & E staining, 20x objective). (C) Liver HL mRNA expression in Ad-L and P injected RAG^−/−LDLR^−/− and LIGHT^−/−LDLR^−/− mice. (D) Liver HL and IL-1β mRNA expression in Ad-L injected LDLR^−/− mice treated with control (C) or clodronate (CL) liposomes. The virus was injected 2 days after clodronate injection. (n = 3; *p<0.05, **p<0.01; for panels A and C: vs. PBS treated mice; for Panel D: vs. control liposome treated mice.).</p

LIGHT-mediated HL regulation in mice is independent of the presence of Kupffer cells.

<p>To deplete Kupffer cells, liposomes containing clodronate were injected through the tail vein into wild type C57BL/6 mice or Tg-LIGHT mice every 5^th day for 14 days. Control liposomes did not contain clodronate. (A) F4/80 staining for liver Kupffer cells in control liposome and clodronate liposome injected Tg-LIGHT mice. (B) Real-time PCR data for HL mRNA expression in the liver of control (C) and clodronate (CL) liposome injected wild type (WT) and Tg-LIGHT mice. (n = 3; *p<0.01 WT vs. Tg-LIGHT).</p

LIGHT and HL expression in Ad-LIGHT transduced primary hepatocytes in vitro.

<p>(A) HL mRNA expression in Ad-LIGHT (Ad-L) and Null adenovirus (Ad-N) infected primary hepatocytes from wild type mice 18, 24 and 36 hours post-infection. The numbers indicate the % decrease in expression in the Ad-LIGHT infected cells relative to the Ad-N infected cells. (B) Total protein from 1×10⁶ Ad-LIGHT infected hepatocytes (in triplicate) at various times post-infection were immunoblotted with anti-mouse LIGHT antibody. Lane 12 is from non-infected hepatocytes and lane 13 is from Tg-LIGHT mouse spleen. (C) Liver HL mRNA expression in Ad-N and Ad-L infected hepatocytes from LDLR^−/− and LTβR^−/−LDLR^−/− mice. (n = 3; *p<0.05, **p<0.01 vs. Ad-N).</p

Evidence for trans-regulation by LIGHT and lack of a role of liver non-parenchymal cells.

<p>(A) HL mRNA expression in hepatocytes from LDLR^−/− (WT) and LTβR^−/−LDLR^−/− mice cocultured with Ad-LIGHT (Ad-L) or Ad-Null (Ad-N) infected FL83B cells. (B) HL mRNA expression in hepatocytes from LTβR^−/−LDLR^−/− mice cocultured with Ad-N or Ad-L infected FL83B cells and Tg-LIGHT mouse liver non-parenchymal cells (NPC). (n = 3; *<0.05 vs. Ad-N infected FL83B coculture).</p

LTβR expression on hepatocytes is sufficient for HL regulation by Ad-LIGHT.

<p>(A) Liver LTβR and HVEM expression in LDLR^−/−, HVEM^−/−LDLR^−/− and hepatocyte-specific knockout of LTβR (H-LTβR^−/−) LDLR^−/− mice. (B and C) Mice were injected with PBS or adenovirus and sacrificed on the 7^th day. Liver HL mRNA expression in Ad-LIGHT (Ad-L) and PBS (P) injected LTβR^−/−LDLR^−/− mice (B) and (H-LTβR^−/−LDLR^−/− and HVEM^−/−LDLR^−/− mice C). (n = 3; **p<0.01 vs. PBS injected mice).</p

Magnetic Field-Activated Sensing of mRNA in Living Cells

Detection of specific mRNA in living cells has attracted significant attention in the past decade. Probes that can be easily delivered into cells and activated at the desired time can contribute to understanding translation, trafficking and degradation of mRNA. Here we report a new strategy termed magnetic field-activated binary deoxyribozyme (MaBiDZ) sensor that enables both efficient delivery and temporal control of mRNA sensing by magnetic field. MaBiDZ uses two species of magnetic beads conjugated with different components of a multicomponent deoxyribozyme (DZ) sensor. The DZ sensor is activated only in the presence of a specific target mRNA and when a magnetic field is applied. Here we demonstrate that MaBiDZ sensor can be internalized in live MCF-7 breast cancer cells and activated by a magnetic field to fluorescently report the presence of specific mRNA, which are cancer biomarkers

YopE_69–77-specific CD8 T cells lacking the capacity to produce TNFα and IFNγ fail to protect mice and control bacterial burden.

<p>TCRβδ-deficient (TCRbdKO) mice were lethally irradiated and reconstituted with 75% TCRβδKO bone marrow cells and 25% of either WT, TNFαKO, IFNγKO, PKO, or TNFαIFNγ DKO bone marrow cells. Six weeks later they were immunized with CT mixed with YopE_69–77 or OVA_257–264 and then challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. (A) Survival. In comparison with OVA_257–264-immunized mice reconstituted with WT T cells (n = 20), the YopE_69–77-immunized chimeric mice reconstituted with WT (n = 25), TNFαKO (n = 17), IFNγKO (n = 19), PKO (n = 8) or TNFαIFNγ DKO (n = 33) T cells all showed significant protection. (B) The percentage of CD8+ T cells that stained positive for MHC class I tetramer K^bYopE_69–77 in PBL on the day before challenge. Solid bar depicts the mean. All groups of chimeric mice that were immunized with YopE_69–77 had significantly increased frequency of K^bYopE_69–77+CD8+ T cells in compared with the chimeric mice immunized with OVA_257–264 (p<0.001). YopE_69–77-immunized chimeric mice reconstituted with TNFαKO T cells had significantly higher frequency of K^bYopE_69–77+CD8+ T cells in compared with YopE_69–77-immunized chimeric mice reconstituted with WT T cells (p<0.01). Data for (A) and (B) are pooled from 6 independent experiments. (C and D) Bacterial burden in lung (C) and liver (D) tissues was measured at day 4 after challenge (Kruskal-Wallis test). Data are pooled from 3 independent experiments. Solid bar depicts median; broken line depicts the limit of detection.</p

Immunization with YopE_69–77 peptide protects mice against <i>Y. pestis</i>.

<p>Wild-type C57BL/6 mice were immunized intranasally with CT adjuvant alone (CT) or CT mixed with YopE_69–77 peptide (YopE) and then challenged intranasally with (A) 20 MLD (2×10⁵ CFU) or (B) 200 MLD (2×10⁶ CFU) <i>Y. pestis</i> strain D27 or (C) 10 MLD (1×10⁴ CFU) <i>Y. pestis</i> strain CO92. In comparison with CT–immunized mice (n = 10–40), YopE_69–77–immunized mice (n = 15–39) exhibited significantly increased survival. Data were pooled from 2–5 independent experiments.</p

TNFα and IFNγ produced by YopE_69–77-specific CD8 T cells have complementary roles during protection against <i>Y. pestis</i>.

<p>(A) Wild-type (WT), TNFα-deficient (TNFaKO) or IFNγ-deficient (IFNgKO) C57BL/6 mice were immunized with CT mixed with YopE_69–77 peptide. WT mice immunized with CT adjuvant only or CT mixed with OVA_257–264 peptide were used as controls. CD8+ splenocytes were then purified and transferred intravenously to naïve WT C57BL/6 mice, which were challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27 the next day. In comparison with mice that received CD8+ T cells from CT-immunized WT mice (n = 50), mice that received CD8+ T cells from YopE_69–77-immunized WT (n = 56), TNFα-deficient (n = 17) and IFNγ-deficient (n = 15) mice were protected against <i>Y. pestis</i> challenge. Notably, mice that received CD8+ T cells from OVA_257–264-immunized WT mice (n = 25) were also protected (p<0.05). Data were pooled from 7 independent experiments. (B) Splenocytes isolated from naïve WT, TNFαKO, IFNγKO, perforin-deficient (PKO) or TNFα/IFNγ-deficient (TNFαIFNγ DKO) mice were transferred to TCRβδ-deficient mice. The mice were then immunized with CT mixed with YopE_69–77. Control mice received naïve WT splenocytes and were immunized with CT mixed with OVA_257–264 peptide. Mice were then challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. In comparison with control mice (n = 15), YopE_69–77 immunized mice that received WT (n = 22), TNFαKO (n = 25), IFNγKO (n = 20), or PKO (n = 11) splenocytes were protected against <i>Y. pestis</i> challenge. YopE_69–77-immunized mice that received TNFαIFNγ DKO splenocytes were also protected (n = 11) but the survival was significantly lower in comparison with the mice received WT, TNFαKO or IFNγKO splenocytes. Data were pooled from 3 independent experiments.</p

Perforin is dispensable for YopE_69–77–specific CD8 T cell-mediated protection against <i>Y. pestis</i> and <i>Y. pseudotuberculosis</i>.

<p>Wild-type (WT) and perforin-deficient (PKO) C57BL/6 mice were immunized intranasally with CT adjuvant alone, or CT mixed with YopE_69–77 or OVA_257–264 peptides and then challenged with (A and B) 20 MLD (2×10⁵ CFU) <i>Y. pestis</i> strain D27 intranasally, (C) 10 MLD (5×10⁹ CFU) <i>Y. pseudotuberculosis</i> strain 32777 intragastrically or (D) 10 MLD (1.2×10² CFU) <i>Y. pseudotuberculosis</i> strain 32777 intravenously. (A) <i>Y. pestis</i> survival data pooled from 3 independent experiments (n = 9–30 mice/group). (B) Day 4 bacterial burden in lung and liver tissues after <i>Y. pestis</i> challenge (Kruskal-Wallis test, compared with CT- or OVA_257–264–immunized PKO or WT mice). Data are pooled from 2 independent experiments (n = 9–11 mice/group). Solid bar depicts median; broken line depicts the limit of detection. (C and D) <i>Y. pseudotuberculosis</i> survival data (n = 6–7 mice/group for CT, n = 10–11 mice/group for YopE). Data were pooled from 2 independent experiments.</p