Study design and immunization schedule.

<p>Study design and immunization schedule.</p

CTLA-4 blockade enhances the proliferative capacity of CD4⁺ and CD8⁺ T cells.

<p>Fresh PBMCs isolated after the second immunization were stained with CFSE and stimulated with SIVgag, env, and pol peptides <i>in vitro</i> for 5 days to determine the proliferative capacity of antigen-specific cells. The proliferative capacity of the CD4⁺(<b>a</b>) and CD8⁺ (<b>b</b>) T cell compartments are shown as stacked group mean responses ± SEM. Statistical differences between groups were determined by a one-way ANOVA with a Tukey post-hoc test.</p

Polyfunctional profile of CD4⁺ T cells.

<p>PBMCs isolated 2 weeks after the fourth immunization were stimulated <i>in vitro</i> with a SIVpol peptide pool mix for 5 hours. Cells were stained for intracellular production of IFN-γ, TNF-α and IL-2 and degranulation by CD107a. The magnitude of the SIVgag (white), env (grey) and pol (black) responses are shown as stacked means ± SEM for each group (<b>a</b>). The percentage of the total functional response that has a CD28⁻CD95⁺ (black bar) or CD28⁺CD95⁺ (white bar) is shown as group means ± SEM (<b>b</b>). Pie charts show the proportion of antigen-specific CD4⁺ T cells that have 4 functions (purple), 3 functions (yellow), 2 functions (green) or 1 function (light blue) (<b>c</b>). The bar graphs depict the absolute frequency of each of the 15 functional combinations for the DNA (red), 4-1BB (blue), CTLA-4 (orange), Combo (green) and Saline (black) groups in response to SIVgag, env, and pol after background subtraction (<b>d</b>).</p

Modulation of the 4-1BB pathway during vaccination results in enhanced control of viral replication following SIVmac251 mucosal challenge.

<p>To test the efficacy of the vaccine-induced immune response the animals were challenged with a high-dose, intrarectal SIVmac251 challenge ten months following the fifth immunization. We then assessed viral loads every week for two months and every month thereafter up to 5 months post-challenge. The viral load data were graphed with the group means shown. (a). Viral loads were compared at peak viremia (2 weeks post challenge), (b.) set point (12 weeks post-challenge) and (c.) during chronic infection (20–24 weeks post-challenge). Statistical analysis was performed by log transforming the viral loads to normalize the data and used in a one-way ANOVA with a Tukey post-hoc test with p values of <0.05 (*) or <0.01 (**) indicated.</p

Maintenance of memory T cells.

<p>PBMCs were collected from immunized macaques at 10 months following a fourth immunization to evaluate memory IFN-γ responses. Cells were then stimulated with SIVmac239 gag (white bars), env (grey bars), and pol (black bars) peptide pools in 18-hour IFN-γ ELISpot assays (<b>a</b>). PBMCs were also labelled with CFSE and stimulated with pooled SIVmac239 gag (white bars), env (grey bars), and pol (black bars) peptides for 5 days. Cells were then stained for phenotypic markers and analyzed by flow cytometry to determine the proliferative response to each antigen (<b>b and c</b>). For both ELISpot and proliferation assays, error bars represent mean responses ± SEM.</p

Polyfunctional profile of CD8⁺ T cells.

<p>PBMCs isolated 2 weeks after the fourth immunization were stimulated <i>in vitro</i> with a SIVpol peptide pool mix for 5 hours. Cells were stained for intracellular production of IFN-γ, TNF-α and IL-2 and degranulation by CD107a. The magnitude of the SIVgag (white), env (grey) and pol (black) responses are shown as stacked means ± SEM for each group (<b>a</b>). The percentage of the total functional response that has a CD28⁻CD95⁺ (black bar) or CD28⁺CD95⁺ (white bar) is shown as group means ± SEM (<b>b</b>). Pie charts show the proportion of antigen-specific CD8⁺ T cells that have 4 functions (purple), 3 functions (yellow), 2 functions (green) or 1 function (light blue) (<b>c</b>). The bar graphs depict the absolute frequency of each of the 15 functional combinations for the DNA (red), 4-1BB (blue), CTLA-4 (orange), Combo (green) and Saline (black) groups in response to SIVgag, env, and pol after background subtraction (<b>d</b>).</p

Modulation of Th1/Th2 responses following DNA vaccination with antibody adjuvants.

<p>An IFN-γ ELISpot was used to measure the Th1 response following each immunization (<b>a</b>). The relative contribution of CD4⁺ and CD8⁺ T cells to the IFN-γ response was measured by ELISpot following CD8⁺ T cell depletion of PBMCs isolated after the fourth immunization (<b>b</b>). The Th2 response was assessed by IL-4 production. ELISpot assay (<b>c</b>). Statistical differences between groups was determined by doing pair-wise Mann-Whitney tests with a Bonferroni adjustment with p values less than 0.01 being significant (** = p<0.01). Pair-wise values: CTLA-4 vs. Saline or DNA (p = 0.002, p = 0.009, respectively); Combo vs. Saline, DNA, or 4-1BB (p = 0.002, p = 0.002, p = 0.009, respectively).</p

The relationship between the cytokine-producing CD4+CD45RO+ T cells and the QuantiFERON Gold In-Tube (QFT) response at baseline.

<p>The relationship between the absolute frequencies of (<b>A</b>) all IFNγ-producing and (<b>B</b>) polyfunctional (IFNγ, IL2 and TNFα) PPD-specific CD4+CD45RO+ T cells and the magnitude of the QFT response are shown. The relationship between the absolute frequencies of cytokine-producing T-cells in response to SEB is shown for comparison (<b>C and D</b>). Statistical analyses were performed by Spearman’s correlation coefficient (rho).</p

QuantiFERON Gold In-Tube (QFT) and Tuberculin Skin Test (TST) responses.

<p>(<b>A</b>) The magnitude of the QFT response and (<b>B</b>) the TST response in QFT consistent positives (n = 21), QFT reverters (n = 21) and QFT negative controls (n = 10) at baseline (BL) and after 1 year. Unpaired and paired analyses was performed with Mann-Whitney U test and Wilcoxon Signed rank test where appropriate. Horizontal lines represent median values.</p

Qualitative and quantitative analysis of PPD-specific CD4+CD45RO+ T-cell responses in QFT consistent positives and QFT reverters.

<p>(<b>A</b>) The relative frequencies of the PPD-specific CD4+CD45RO+ T-cell subsets in QFT consistent positives (red symbols) and QFT reverters (blue symbols) at baseline (filled) (n = 16, n = 7) and after 1 year (open) (n = 6, n = 6). The subsets are defined on the basis of IFNγ, TNFα and/or IL-2 production. Since relative frequencies are normalized data, un-paired T-test with Welch’s correction was applied for statistical analyses and horizontal lines represent mean values. (<b>B</b>) The absolute frequencies of PPD-specific CD4+CD45RO+ T- in QFT consistent positives (red symbols) and QFT reverters (blue symbols) at baseline (filled) (n = 16, n = 7) and after 1 year (open) (n = 6, n = 6). As the data are not normally distributed, statistical analyses were performed by the Mann-Whitney U test and horizontal lines represent median values. (<b>C</b>) Flowcytometric plots of the functional profile of PPD-specific CD4+CD45RO+ T-cells compared with negative (PBS) and positive (SEB) control in one representative QFT consistent positive subject are shown.</p