Peptide-specific CD8 T cell responses against the HIV-1 Gag antigen in mice immunized with Gag or with the combination Gag and Tat

After 3 immunizations, fresh splenocytes were pooled and purified, then CD8+ T cells were tested by IFNγ Elispot assay after stimulation with the indicated 15 mers (panel A), or with the indicated 8–10 mers derived from the above 15 mers (panel B). Results from mice immunized with Gag (empty bars) and from mice immunized with Gag and Tat (filled bars) are expressed as SFU ± SD/10cells. The mean +/- SD of three independent experiments is shown.<p><b>Copyright information:</b></p><p>Taken from "Identification of new HIV-1 Gag-specific cytotoxic T lymphocyte responses in BALB/c mice"</p><p>http://www.virologyj.com/content/5/1/81</p><p>Virology Journal 2008;5():81-81.</p><p>Published online 14 Jul 2008</p><p>PMCID:PMC2481256.</p><p></p

Tat-mediated CD8⁺ T cell activation.

<p>(<b>A</b>) Fresh splenocytes from C57BL/6 mice were co-cultured, in the presence or absence of 1µg/ml of Tat, with autologous splenocytes previously loaded with the SSI peptide epitope. After 8 days, cells were assayed in IFNγ Elispot. One representative experiment out of five is shown. (<b>B</b>) Splenocytes were purified from HSV1-infected C57BL/6 mice at day 8 post-infection and assayed in IFNγ Elispot against the SSI epitope in the presence or absence of 1 µg/ml of Tat. One representative experiment out of five is shown. (<b>C</b>) Balb/c mice (3 per groups) were injected with 5 µg of Gag alone or in combination with 5 µg of Tat. Ten days after vaccination mice were sacrificed and fresh splenocytes assayed in IFNγ Elispot against the indicated Gag-derived T cell peptide epitopes (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077746#pone.0077746.s002" target="_blank">Table S1</a>). One representative experiment out of three is shown.</p

Tat modulates the kinetics and the magnitude of CTL responses.

<p>Splenocytes were purified from control and Tat-treated HSV1-infected C57/BL6 mice at days 4, 5, 6, 7, 8, 10 and 13 post-infection. (<b>A</b>) Percentage of SSI-specific CD8⁺ T cells detected by dextramer staining. Data are presented as mean ± SEM of 5 mice per group. One representative experiment out of three is shown. (<b>B</b>) Kinetics of SSI- and QTF-specific cellular responses detected by IFNγ Elispot on fresh splenocytes. Data are presented as mean ± SEM of 5 mice per group (left panel). Expansion and contraction were normalized, for each group, to values detected at day 7 (right panel). One representative experiment out of three is shown. (<b>C</b>) SSI- and QTF- specific IFNγ responses at days 5 and 6 post-infection. (<b>D</b>) SSI- and QTF- specific IFNγ responses at day 7 post-infection. (<b>E</b>) SSI- and QTF-specific IFNγ responses at day 13 post-infection. For statistical analysis two-tailed Mann Whitney test was used. *P<0.05. </p

Tat does not contribute to the control of HSV1 acute infection.

<p>Control and Tat-treated HSV1-infected C57/BL6 mice were checked daily for the appearance of disease signs. (<b>A</b>) Mean of disease scores ± SEM of 20 mice per group is shown. For statistical analysis two-tailed Mann Whitney test was used. **P<0.01. (<b>B</b>) Probability of developing disease signs is shown for each group. Figure represents Kaplan-Meier estimation of the probability of clinical manifestations. For statistical analysis Log rank test was used. One representative experiment out of three is shown.</p

Tat administered at the time of antigen-priming favors an effector memory phenotype.

<p>Control and Tat-treated mice were infected with HSV1 wt (<b>A</b> and <b>B</b>) or with replicative-defective HSV1 (<b>C</b> and <b>D</b>) and sacrificed at days 70 post-infection. (<b>A</b> and <b>C</b>) Percentage of SSI-specific CD8⁺ T cells detected by dextramer staining. (<b>B</b> and <b>D</b>) CD62L expression was measured by flow cytometry on SSI-specific CD8⁺ T cells. Data are presented as mean ± SEM of 5 mice per group. For statistical analysis two-tailed Mann Whitney test was used. *P<0.05. One representative experiment out of three is shown.</p

Tat administered at the time of antigen-priming favors a Th1 profile of the humoral response.

<p>Blood samples from control and Tat-treated HSV1-infected mice were collected and the presence of anti-HSV1 antibodies was detected by ELISA assay. (<b>A</b>) Anti-HSV1 IgG1 and IgG2a were measured at days 20 (left) and 70 (right) post-infection. (<b>B</b>) Total anti-HSV1 IgG were measured at days 20 (left) and 70 (right) post-infection. Data are presented as mean ± SEM of 5 mice per group. For statistical analysis two-tailed Mann Whitney test was used. *P<0.05. One representative experiment out of three is shown.</p

Tat does not activate bystander T cells.

<p>Control and Tat-treated HSV1-infected C57/BL6 mice were sacrificed at days 4, 6, 8, 10 and 13 post-infection. CD8⁺ (<b>A</b>), CD4⁺ (<b>C</b>) and B (<b>D</b>) lymphocytes numbers were measured by flow cytometry. Data are presented as mean ± SEM of 5 mice per group. For statistical analysis two-tailed Mann Whitney test was used. *P<0.05. One representative experiment out of three is shown. (<b>B</b>) CD95 expression was measured by flow cytometry on CD8⁺ T cells. One representative experiment out of five is shown.</p

Additional file 2: of Association between different anti-Tat antibody isotypes and HIV disease progression: data from an African cohort

magnitude of anti-Tat humoral responses: (A) Anti-clade B and C Tat IgG, IgA or IgM titers were determined in anti-Tat positive sera and displayed for every donor. (B) Heatmap showing, for single donors, positivity (black) or negativity (grey) towards clade C or clade B Tat for each isotype. (C) Optical density (O.D.) from screening ELISA tests are shown for each donor after cut-off subtraction. Cut-off values were included in the following ranges: 0.128 ± 0.029 for anti-clade C Tat IgG; 0.1245 ± 0.042 for anti-clade B Tat IgG; 0.1177 ± 0.022 for anti-clade C Tat IgA; 0.1387 ± 0.031 for anti-clade C Tat IgA; 0.1963 ± 0.060 for anti-clade C Tat IgM; 0.370 ± 0.086 for anti-clade B Tat IgM. (JPG 154 kb

Additional file 3: of Association between different anti-Tat antibody isotypes and HIV disease progression: data from an African cohort

Frequencies of CD4+ and CD8+ T cells expressing activation and maturation markers in the peripheral blood: Shown are representative plots demonstrating the gating strategy for the expression of activation (CD38 and HLA-DR; left panel) and maturation (CD27 and CD45RO; right panel) markers on CD4+ (upper panels) and CD8+ (lower panels) T cells. (JPG 316 kb

Effects of RAMB treatment on the levels of polyubiquitinated proteins in HeLa cells.

<p><i>Left panel:</i> immunoblot analysis of ubiquitinated proteins in HeLa cells after 6 hours exposure with or without 10 µM RAMBs. Bortezomib was used as positive control. Equal protein loading in each lane was verified by using an antibody against GAPDH. <i>Right panel:</i> Quantification of the Ubiquitin/GAPDH ratios.</p