Vaccination with SLA-CpG-DCs induces a CXCL10 mediated CD8⁺ T cell response in parasitized mice.

<p>(<b>A</b>) Spenocytes (1×10⁶) were isolated from spleen of infected, differently vaccinated and CXCL10 depleted vaccinated mice after 0, 7, 14, 28, 35 and 56 days after infection. Splenocytes were then harvested, stimulated for 4 h with SLA (10 µg/ml), and then stained for IFN-γ and CD8. Then cells were analyzed for expression of CD8 and IFN-γ on a flow cytometer. The values shown (plotted on a log10 scale) reflect the average number of antigen-specific IFN-γ⁺CD8⁺ T cells expressed as a percentage of total CD8⁺ T cells present in the spleens. Results are from 3 independent experiments and represent the mean values ± standard errors of the means for 5 animals per group per time point. (<b>B</b>) Increased CD8⁺ T Cell Proliferation in CD8⁺ T cells purified from SLA-CpG-DC vaccinated parasitized mice. CD8⁺ T cells (1×10⁶) were purified from spleens of infected, differently vaccinated mice and CXCL10 depleted vaccinated mice (methods) after 4 weeks post infection and were stimulated in presence of ConA (2.5 µg/ml) or in presence or absence of SLA (10 µg/ml) and after 72 h incubation, proliferation was measured by ³H thymidine incorporation. Values and bars represent mean CPM and the standard deviation and are representative of three independent experiments. **<i>P</i><.001, compared to unstimulated controls. CPM: counts per minute (<b>C</b>) IFN-γ production was enhanced in CD8⁺ T cells purified from SLA-CpG-DC vaccinated parasitized mice. CD8⁺ T cells (1×10⁶) were purified from spleens of differently vaccinated mice (methods) after 4 weeks post infection and plated with T-depleted, mitomycin C-treated syngeneic APCs (5×10⁵). The cells were then stimulated in presence or absence of SLA (10 µg/ml) for 72 h. Samples were assayed in triplicate by ELISA. Values and bars represent mean CPM and the standard deviation. The experiments were repeated thrice more with similar results. **<i>P</i><.001, compared to unstimulated controls. (<b>D</b>) Contribution of CD8⁺ T cells to SLA-CpG-DCs mediated vaccination against <i>Leishmania donovani.</i> CD8⁺ T cells were depleted by one intraperitoneal (i.p.) injection of CD8 depleting antibody (100 µg) 1 day before the vaccination. In one group of similarly vaccinated and infected mice a control IgG Ab was injected. Mice were subsequently immunized i.v. with SLA-CpG-DCs and were challenged with <i>L. donovani</i> promastigotes 7 days later. Control mice were treated with PBS. Mice were sacrificed on day 28 after infection. Levels of parasite burden in liver and spleen samples were determined by stamp-smear method and expressed in Leishman Donovan Units (LDU). Results are from 3 independent experiments and represent the mean values ± standard errors of the means for 3 animals per group per time point. **<i>P</i><.001 compared to SLA-CpG-treated infected mice.</p

CXCL10 Is Critical for the Generation of Protective CD8 T Cell Response Induced by Antigen Pulsed CpG-ODN Activated Dendritic Cells

<div><p>The visceral form of leishmaniasis is the most severe form of the disease and of particular concern due to the emerging problem of HIV/visceral leishmaniasis (VL) co-infection in the tropics. Till date miltefosine, amphotericin B and pentavalent antimony compounds remain the main treatment regimens for leishmaniasis. However, because of severe side effects, there is an urgent need for alternative improved therapies to combat this dreaded disease. In the present study, we have used the murine model of leishmaniasis to evaluate the potential role played by soluble leishmanial antigen (SLA) pulsed-CpG-ODN stimulated dendritic cells (SLA-CpG-DCs) in restricting the intracellular leishmanial growth. We found that mice vaccinated with a single dose of SLA-pulsed DC stimulated by CpG-ODN were protected against a subsequent leishmanial challenge and had a dramatic reduction in parasite burden along with the generation of parasite specific cytotoxic T lymphocytes. Moreover, we demonstrate that the induction of protective immunity conferred by SLA-CpG-DCs depends entirely on the CXC chemokine IFN-γ-inducible protein 10 (CXCL10; IP-10). CXCL10 is directly involved in the generation of a parasite specific CD8⁺ T cell-mediated immune response. We observed significant reduction of CD8⁺ T cells in mice depleted of CXCL10 suggesting a direct role of CXCL10 in the generation of CD8⁺ T cells in SLA-CpG-DCs vaccinated mice. CXCL10 also contributed towards the generation of perforin and granzyme B, two important cytolytic mediators of CD8⁺ T cells, following SLA-CpG-DCs vaccination. Together, these findings strongly demonstrate that CXCL10 is critical for rendering a protective cellular immunity during SLA-CpG-DC vaccination that confers protection against <em>Leishmania donovani</em> infection.</p> </div

CXCL10 depletion reduces the cytotoxicity of CD8⁺ T cells in SLA-CpG-DCs vaccinated parasitized mice.

<p>(<b>A, B</b>) CD8⁺ T cells (1×10⁶) were purified from spleens of infected, SLA-CpG-DCs vaccinated and CXCL10 depleted vaccinated mice (methods) after 4 weeks post infection, harvested and stimulated for 4 h with SLA (10 µg/ml), followed by fixation and staining for Granzyme B-PE (<b>A</b>) and Perforin-FITC (<b>B</b>) as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048727#s2" target="_blank">materials and methods</a>. The data was analyzed by flow cytometry. These data were from one of three experiments conducted in the same way with similar results. (<b>C, D</b>) A separate set of CD8⁺ T cells (2×10⁶), of the above mentioned groups was collected in Trizol for mRNA extraction and subjected to RT (Reverse Transcriptase) PCR as described in Methods. Expression of granzyme B (<b>C</b>) and perforin (<b>D</b>) from CD8⁺ T cells was observed in upper panel; Group 1: Control, Group 2: Infection, Group 3: Infection+ SLA-CpG-DCs and Group 4: Infection+ SLA-CpG-DCs and Group+ CXCL10 mAbs. Lower panel shows GAPDH expression levels. Band intensities were analyzed by densitometry. Results are representative of three experiments conducted in the same way with similar results.</p

Immunophenotyping of BMDCs.

<p>(<b>A</b>) Purity of dendritic cells. BALB/c mice bone marrow cells were cultured in the presence of GM-CSF and IL-4 for 7 days. Then these bone marrow-derived DCs (BMDCs) were washed and adhered for 3 h. The adherent cells were stained with CD11c-PE and analyzed on a flow cytometer. (<b>B</b>) Purified DCs were stained with CD80-PE, CD86-PE and MHC class II-FITC antibodies to detect their maturation levels following CpG-ODN (10 µg/ml) stimulation. The data show histograms of cell number against fluorescence intensity and are representative of three experiments. Solid areas indicate staining with isotype matched antibodies. Lines show staining of stimulated and un-stimulated DCs with the indicated mAbs. (<b>C</b>) DCs (1×10⁶ cells /ml) were stimulated with SLA (10 µg/ml) or CpG-ODN (10 µg/ml) alone or in combinations for 24 hours. In some experimental sets, DCs were transfected with TLR9-specific siRNA or control siRNA. Briefly, DCs (10⁶ cells) were transfected with TLR9 siRNA at a final concentration of 100 nM, using transfection reagent Oligofectamine (Invitrogen, Carlsbad, CA, USA) as per manufacturer’s instructions. The DCs were incubated with the transfection complexes in RPMI without serum for 6 h followed by another 18 h with 10% FBS. Control cultures were transfected using scrambled TLR9 siRNA (100 nM). Cells were then washed, and treated with SLA and CpG-ODN. Cell supernatants were then obtained for detecting CXCL10 levels by ELISA. Data are means ± standard deviations of values from 3 independent experiments conducted in the same way that yielded similar results. **<i>P</i><.001, compared to unstimulated control. n.d. not detectable.</p

CD8⁺ T cell cytotoxicity. CD8⁺ T cells purified from SLA-CpG-DCs vaccinated parasitized mice 28 days after infection were co-incubated with autologous uninfected (I) and <i>Leishmania</i>-infected macrophages (II) in a 10∶1 ratio.

<p>In another set, CD8⁺ T cells were purified from CXCL10 depleted SLA-CpG-DCs vaccinated parasitized mice 28 days after infection and co-incubated with autologous <i>Leishmania</i>-infected macrophages in a 10∶1 ratio (<b>III</b>). After 4 h, the cells were harvested and stained with anti-CD14-FITC antibodies to select the target macrophages (<b>A</b>). The dot plots were derived from the gated events based on the region encircling positive cells. This CD14-FITC positive population were analyzed for propidium iodide staining to detect killed macrophages (<b>B</b>). M1: Apoptotic peak, M2: G1 peak, M3: S peak and M4: G2+M peak. The data was analyzed on a flow cytometer (FACS Calibur), using the Cell Quest program. These data were from one of three experiments conducted in the same way with similar results. The error bars represent mean ± SD. **<i>P</i><.001, compared to SLA-CpG-DC vaccinated parasitized mice.</p

SLA-CpG-DCs vaccination induces enhanced production of IgG2a.

<p>Leishmania specific Ab responses in infected and differently vaccinated <i>L. donovani</i>-infected BALB/c mice were studied. Sera from infected and differently vaccinated BALB/c mice were collected at 28 Days and analyzed for Leishmania Ag-specific anti-IgG1 and anti-IgG2a levels by ELISA. Results are from 3 independent experiments and represent the mean values ± standard errors of the means for 5 animals per group. **<i>P</i><.001, compared to infected mice.</p

Effect of <i>in vivo</i> treatment with SLA-CpG-DCs on the parasite load in liver and spleen of Leishmania donovani–infected BALB/c mice.

<p>Mice were either vaccinated with SLA-pulsed, SLA+ CpG-ODN pulsed, SLA+ control-CpG-ODN pulsed, only CpG-ODN-pulsed DCs or either phosphate buffered saline (PBS; control) followed by intravenous infection with 1×10⁷ stationary phase <i>Leishmania donovani</i> promastigotes after 7 days. Mice were sacrificed on days 1, 7, 14, 28, and 56 after infection. Levels of parasite burden in liver (A) and spleen (B) samples were determined by stamp-smear method and expressed in Leishman Donovan Units (LDU). Results are from 3 independent experiments and represent the mean values ± standard errors of the means for 5 animals per group per time point. **<i>P</i><.001 and *<i>P</i><.005, compared to infected mice. (C) Cured mice after 5 weeks of vaccination (4 weeks of infection) along with age-matched controls were re-infected with similar dose of <i>Leishmania donovani</i> and at 12 weeks of primary infection, were sacrificed and liver and spleen parasitic loads were determined by stamp-smear method and expressed as Leishman Donovan Units (LDU). Results are from 3 independent experiments and represent the mean values ± standard errors of the means for 5 animals per group per time point. **<i>P</i><.001, compared to infected mice.</p

Regulatory effect of PKCδ and PLD1 on aSMase mediated ceramide generation.

<p>(A) B16F10 cells overexpressing full-length mouse PKCδ (PKCδOV) or empty vector (EV) were treated with Fumonisin B1(FB1) (10 uM) and Imipramine (10 uM) respectively for 1 hour. The transfected cells were washed and after 24 hour of incubation were stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody, and analyzed by flow cytometry for ceramide (FL1) expression. Representative data exhibited were from three independent experiments. (B) The transfectants of B16F10 cells overexpressing PKCδ (PKCδOV) or the empty vector (EV) were treated with FB1 (10 uM) and Imipramine (10 uM) for 1 hour. The transfected cells were washed and after 24 hours of incubation the whole cell lysates were subjected to SDS-PAGE and Western blot analysis to study the expression of pAKT and total AKT. Data represented here are from one of three independent experiments. (C) Similarly, B16F10 cells transfected with empty vector (EV) and full-length PKCδ (PKCδOV) were treated with FB1 (10 uM) and Imipramine (10 uM) as mentioned previously and expression of PLD1 was analysed by western blotting. Data represented here are from one of three independent experiments. GAPDH was used as a reference.</p

Modulatory role of PKCα and PKCδ on the expression of pAKT and ceramide levels.

<p>(A) B16F10 cells transfected with EV, full-length mouse PKCαOV, PKCδOV and siRNA oligonucleotides specific to PKCα (PKCα siRNA), PKCδ (PKCδ siRNA) or the control siRNA were collected and the whole cell lysates prepared from transfected cells and the expression of pAKT or AKT respectively were analyzed with respect to GAPDH by Western blotting. Representative blots from three independent experiments were given. (B) The transfected cells comprising empty vector (EV), overexpressed PKCα (PKCαOV), PKCδ (PKCδOV) or siRNAs corresponding to PKCα (PKCα siRNA), PKCδ (PKCδ siRNA) and control siRNA were stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody, and analyzed by flow cytometry for ceramide (FL1) expression. (C) B16F10 transfectants overexpressing full-length mouse PKCα (PKCOV), PKCδ (PKCδOV) or empty vector (EV) were transfected with PLD1 siRNA as mentioned in materials and methods and harvested for 24 hours. The expression of pAKT and AKT was analysed in whole cell lysates by Western blotting. (D) Similarly full-length mouse PKCα (PKCαOV), PKCδ (PKCδOV) or empty vector (EV) transfected B16F10 melanoma cells treated with PLD1 siRNA and subsequently stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody. Ceramide (FL1) expression was analysed by flow cytometry. Data represented here are from one of three independent experiments.</p

Regulatory effect of PKCα and PKCδ on caspase cascade and apoptosis.

<p>(A) Whole cell lysates of B16F10 cells, transfected with empty vector (EV), full-length mouse PKCα (PKCαOV) and PKCδ (PKCδOV) plasmid as mentioned in materials and methods were probed by Western blotting to study the expression of pro Caspase 3, Caspase 3, pro Caspase 8, and Caspase 8. Representative data from three independent experiments was shown compared to GAPDH. (B) Whole cell lysates of B16F10 cells, transfected with empty vector (EV), full-length mouse PKCα (PKCαOV) and PKCδ (PKCδOV) plasmid were probed by Western blotting to study the expression of Bax and Bcl2 respectively. The ratio of Bax (proapoptotic)/Bcl2 (antiapoptotic) were represented on a log scale. (C) Similarly B16F10 cells, transfected with control vector (EV), full-length mouse PKCα (PKCαOV) and PKCδ (PKCδOV) plasmid as mentioned in materials and methods were stained with Annexin-V FITC and PI and analyzed by flow cytometry. Data represented here are from one of three independent experiments. (D) B16F10 cells, transfected with control vector (EV), full-length mouse PKCα (PKCαOV) and PKCδ (PKCδOV) plasmid as mentioned in materials and methods were stained with FITC-BrdU for tunnel assay and analysed by immunofluorescence microscopy.</p