Additional file 1: of Exosomes from patients with septic shock convey miRNAs related to inflammation and cell cycle regulation: new signaling pathways in sepsis?

Supplementary methods and results. (PDF 4801 kb

Summary of bioinformatic analyses for patients at T3.

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Activation of antigen-specific IFN-γ-producing CD8+ T cell precursors in mice immunized with pIRES I or pIRES II.

<p>(A-B) Spleen cells from BALB/c mice spleen cells were stimulated with the MHC-I-restricted p24-specific peptide, and the p24-specific IFN-γ-producing CD8⁺ T cells were detected by intracellular cytokine staining (A) or ELISPOT assay (B). (C–D) Spleen cells from C57BL/6 mice were stimulated with the MHC-I-restricted E7-specific peptide, and the E7-specific IFN-γ-producing CD8⁺ T cells were detected by IFN-γ intracellular staining (C) or ELISPOT assay (D). Mice were i.m. immunized with three doses of the DNA vaccines with one week intervals between doses (100 µg/dose). The CD8⁺ T-cell responses were analyzed two weeks after the last dose. *p<0.05. Data represent the compilation of two independent experiments with four mice per immunization group (n = 8) and results expressed by each animal analyzed. pIRES is the empty vector used as immunization control.</p

Construction of bicistronic DNA vaccines encoding HPV, HIV and HSV antigens for expression in mammalian cells.

<p>(A) Schematic linear representation of the trivalent DNA vaccines. pIRES I and pIRES II contain gDp24 and gDE7 chimeric gene fusions, which are inverted with regard to the CMV promoter and IRES sequence. The empty vector pIRES Ø was used as a control. The nucleotide numbers corresponding to the IRES sequence and the cloned chimeric genes are indicated. (B) In vitro expression of the chimeric proteins encoded by pIRES I (left panels) and pIRES II (right panels). Non-permeabilized pIRES I- or pIRES II-transfected COS-7 cells were labeled with antigen-specific antibodies for the simultaneous detection of the HSV-1 protein gD and the HIV-1 protein p24 or the HPV-16 oncoprotein E7. Green, gD; red, p24 or E7; yellow, co-localization of gD with p24 or E7; blue, DAPI nuclear staining.</p

Induction of <i>in vivo</i> E7- and p24-specific cytolytic CD8⁺ T cell responses in immunized mice.

<p>(A–B) <i>In vivo</i> antigen-specific CD8⁺ T cell-dependent cytotoxic responses in the vaccinated mice was measured two weeks after the last immunization dose. Spleen cells from BALB/c or C57BL/6 mice were labeled with CFSE and pulsed with synthetic peptides representing the immunodominant MHC-I-restricted epitopes of p24 (A) or E7 (B). Data shown in A and B represent the compilation of two independent experiments, encompassing four and five mice per group (n = 9) with results based on the response of each animal. (C) The protective immunity elicited in BALB/c mice immunized with pIRES I or pIRES II was measured after challenge with a recombinant vaccinia virus expressing the HIV-1 protein Gag. Female BALB/c mice were challenged with 2×10⁶ P.F.U. of rVV-Gag, and 5 days later, the level of viable vaccinia virus in the ovaries was determined after titration in Vero cells. Data shown in C represent the compilation of two independent experiments carried out with pooled samples from five mice per group. *p<i><</i>0.05. (D) Prophylactic and (E) therapeutic anti-tumor immunity in C57BL/6 mice immunized with pIRES I or PIRES II. The prophylactic anti-tumor effects were determined in five vaccinated female mice after the s.c. transplantation of 7.5×10⁴TC-1 cells two weeks after the last vaccination. The therapeutic anti-tumor effects induced by the vaccines were determined after transplantation of 7.5×10⁴TC-1 cells one day before the administration of the first vaccine dose. Data shown in D and E represent the compilation of two independent experiments, with five mice per group. The survival curves D and E raised <i>p</i> values of 0.0001 and 0.0006, respectively, in the Logrank test for trend. pIRES is the empty vector used as immunization control.</p

Impaired immunity of specific CD8⁺ T cells upon genetic vaccination of WT mice reconstituted with immunoproteasome-deficient bone marrow.

<p>(a) Experiment design: WT mice were irradiated and reconstituted with WT (WT-WT) or TKO (TKO-WT) bone marrow. After 8 weeks, chimeric mice were vaccinated with adenovirus 5 expressing beta-galactosidase (Adβ-gal) or ASP-2 (AdASP-2). Twenty days later, the response of CD8⁺ T cells was assessed in the spleen. (b) gMFI (geometric mean of fluorescence intensity) of H-2K^b staining of CD11c⁺ splenic cells from WT-WT and TKO-WT chimeras. (c) Staining of TCR Vβ chains gated in CD8+ T cells from WT-WT and TKO-WT chimeras genetically immunized with Adβ-gal. (d) Representative samples and (e) total numbers of specific CD8⁺ T cells stained with H-2K^b-VNHRFTLV pentamers. (f) Representative samples and (g) total numbers of CD8⁺ splenic cells positively stained with anti-TNF and/or anti-IFN-γ after <i>ex vivo</i> restimulation with the peptide VNHRFTLV corresponding to the immunodominant MHC class I-restricted epitope from ASP-2. (h) Combination of cytokines stained in responder CD8⁺ T cells from spleens of AdASP-2-vaccinated mice restimulated <i>ex vivo</i> with VNHRFTLV peptide. Results are shown as individual values and as the mean ± SEM for each group (n = 3). One representative of two independent experiments is shown. Asterisks indicate that the values observed for TKO mice were significantly lower than those for WT mice (**P<0.01 ***P<0.001 ****P<0.0001).</p

Immunoproteasome-deficient mice present impaired immunity of specific CD8⁺ T cells upon genetic vaccination against <i>T</i>. <i>cruzi</i>.

<p>(a) Experiment design: WT and TKO mice were primed with empty plasmid DNA (pcDNA3) or a vector expressing ASP-2 (pIgCl9) and boosted after 21 days with adenovirus 5 expressing beta-galactosidase (Adβ-gal) or ASP-2 (AdASP-2), respectively. Fifteen days later, the response of CD8⁺ T cells was assessed in the spleen. (b) Total numbers of CD8⁺ CD44^high CD62L^low cells. (c) Representative samples and (d) total numbers of specific CD8⁺ T cells stained with H-2K^b-VNHRFTLV pentamers. (e) Representative samples and (f) numbers of CD8⁺ splenic cells positively stained for TNF and/or IFN-γ after <i>ex vivo</i> restimulation with the peptide VNHRFTLV corresponding to the immunodominant <i>T</i>. <i>cruzi</i> MHC class I-restricted epitope from ASP-2. (g) Combination of cytokines stained in responder CD8⁺ T cells from spleens of ASP-2-vaccinated mice restimulated <i>ex vivo</i> with VNHRFTLV peptide. Results are shown as individual values and as the mean ± SEM for each group (n = 4). One representative of two independent experiments is shown. Asterisks indicate that the values observed for TKO mice were significantly lower than those for WT mice (*P<0.05 ****P<0.0001).</p

Immunoproteasome-deficient mice present impaired immunity of specific CD8⁺ T cells upon infection with <i>T</i>. <i>cruzi</i>.

<p>(a) Experiment design: WT and TKO mice were infected <i>s</i>.<i>c</i>. with 10⁴ <i>T</i>. <i>cruzi</i> parasites (Y strain) or left uninfected. Twenty days later, the response of CD8⁺ T cells was assessed in the spleen. (b) Total numbers of CD8⁺ CD44^high CD62L^low cells. (c) Representative samples and (d) total numbers of specific CD8⁺ T cells stained with H-2K^b-VNHRFTLV pentamers. (e) Representative samples and (f) numbers of CD8⁺ splenic cells positively stained with anti-TNF and/or anti-IFN-γ after <i>ex vivo</i> restimulation with the indicated peptides corresponding to known or hypothetical <i>T</i>. <i>cruzi</i> MHC class I-restricted epitopes. (g) Combination of cytokines stained in responder CD8⁺ T cells from spleens of <i>T</i>. <i>cruzi</i>-infected mice restimulated <i>ex vivo</i> with VNHRFTLV peptide. Results are shown as individual values and as the mean ± SEM for each group (n = 3). One representative of two independent experiments is shown. Asterisks indicate that the values observed for TKO mice were significantly lower than those for WT mice (**P<0.01 ****P<0.0001).</p

Mice lacking immunoproteasomes have diminished expression of MHC class I molecules and reduced numbers of CD8⁺ T cells.

<p>WT and TKO mice were infected <i>s</i>.<i>c</i>. with 10⁴ <i>T</i>. <i>cruzi</i> parasites (Y strain) or left uninfected. Twenty days later, spleen cells were collected and stained for surface markers. (a) gMFI (geometric mean of fluorescence intensity) of H-2K^b and (b) I-A^b stainings are shown after gating in CD11c⁺ I-A^b+ CD3^- CD19^- cells. Total numbers of CD8⁺ and CD4⁺ T cells in the spleen of these animals are shown in (c) and (d), respectively. Results are shown as individual values and as the mean ± SEM for each group (n = 3). One representative of at least two independent experiments is shown. Asterisks indicate that the values observed for TKO mice were significantly different than those for WT mice (*P<0.05 **P<0.01 ***P<0.001).</p

Impaired immunity of specific CD8⁺ T cells upon infection with <i>T</i>. <i>cruzi</i> of WT mice reconstituted with immunoproteasome-deficient bone marrow.

<p>(a) Experiment design: WT mice were irradiated and reconstituted with WT (WT-WT) or TKO (TKO-WT) bone marrow. After 8 weeks, the chimeric animals were infected <i>s</i>.<i>c</i>. with 10⁴ <i>T</i>. <i>cruzi</i> parasites or left uninfected. Twenty days later, the response of CD8⁺ T cells was assessed in the spleen. (b) gMFI (geometric mean of fluorescence intensity) of H-2K^b staining of CD11c⁺ splenic cells from WT-WT and TKO-WT chimeras. (c) Staining of TCR Vβ chains gated in CD8+ T cells from <i>naïve</i> WT-WT and TKO-WT chimeras. (d) Representative samples and (e) total numbers of specific CD8⁺ T cells stained with H-2K^b-VNHRFTLV pentamers. (f) Representative samples and (g) total numbers of CD8⁺ splenic cells positively stained with anti-TNF and/or anti-IFN-γ after <i>ex vivo</i> restimulation with the indicated peptides corresponding to known or hypothetical <i>T</i>. <i>cruzi</i> MHC class I-restricted epitopes. (h) Combination of cytokines stained in responder CD8⁺ T cells from spleens of <i>T</i>. <i>cruzi</i>-infected mice restimulated <i>ex vivo</i> with VNHRFTLV peptide. Results are shown as individual values and as the mean ± SEM for each group (n = 3). One representative of two independent experiments is shown. Asterisks indicate that the values observed for TKO mice were significantly lower than those for WT mice (*P<0.05 **P<0.01 ***P<0.001 ****P<0.0001).</p