Frequency of CD4^<b>+</b> T_CM and CD4⁺T_E/EM cells producing IL-2 upon recall with CSP peptides.

<p>PBMC obtained at the indicated time points from RTS,S-immunized subjects were incubated as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g002" target="_blank">Fig. 2</a> with CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cells were then analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>. The values are plotted as the frequency of IL-2-producing CD4⁺ T_CM cells (open bars) and IL-2-producing CD4⁺ T_E/EM cells (closed bars) for protected (right-hand panels) and non-protected (left-hand panels) subjects. Error bars indicate 95% confidence intervals. *p<0.05; **p<0.01; ***p<0.001.</p

CSP-specific IL-2⁺ T_E/EM and T_CM CD4⁺ T cells correlate with anti-CSP R region Ab titers.

<p>The figures display the log base 10 of the IgG level in micrograms per milliliter versus log of the percent of T cells expressing IL-2 as assayed by flow cytometry. Individuals who were protected from malaria by vaccination are noted with filled circles, while those not protected by open circles. The figure also shows the Spearman's correlation between the log IgG and log percent cells for the combined protected and non-protected individuals as well as a trend line.</p

Frequency of CD4^<b>+</b> T_CM and CD4^<b>+</b>T_EM cells producing IFN-γ upon recall with CSP peptides.

<p>PBMC were obtained at the indicated time points from RTS,S-immunized subjects and were incubated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g003" target="_blank">Fig. 3</a> with CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cells were then analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>. The resulting values are plotted as the % of [A] IFN-γ producing CD4⁺ T_CM cells and [B] IFN-γ producing CD4⁺ T_E/EM cells for protected (closed symbol) and non-protected (open symbol) subjects. Statistically significant differences between pre-immune and post-vaccination time points are indicated at the top of the graph. Statistically significant differences between values for protected and non-protected subjects are as indicated within the body of the graph. *p<0.05; **p<0.01; ***p<0.001.</p

Frequency of CD4⁺T_CM and T_E/EM cells producing TNF-α upon recall with CSP peptides.

<p>PBMC were obtained at the indicated time points from RTS,S-immunized subjects and were incubated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g003" target="_blank">Fig. 3</a> with CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cells were then analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>. The values are plotted as [A] the % TNF-α-producing CD4⁺ T_CM cells and [B] the % TNF-α-producing CD4⁺ T_E/EM cells for protected (closed symbol) and non-protected (open symbol) subjects. Statistically significant differences between pre-immune and post-vaccination time points are indicated at the top of the graph. Statistically significant differences between values for protected and non-protected subjects are as indicated within the body of the graph. *p<0.05; **p<0.01; ***p<0.001.</p

Number of cells per subset on the DOC.

a<p>per µl of peripheral blood.</p>b<p>mean ± SEM.</p>c<p>Mann-Whitney <i>U</i>-test.</p

Identification of RTS,S vaccine-induced CSP-specific CD4⁺ T cell memory subsets.

<p>PBMCs from an RTS,S-immunized volunteer were stimulated in vitro for 18 h with anti-CD28 and anti-CD49d plus a pool of 2 long contiguous CSP peptides in the presence of PE-conjugated anti-CCR7 Ab. Cells were harvested and surface-labeled for CD3, CD4, CD8, CD45RO and UV viability dye and then stained intra-cellularly for IL-2, TNF-α and IFN-γ. Cells were acquired on an LSR-II flow cytometer and analyzed using FlowJo. (A) Gating strategy used to identify CD4⁺CD45RO⁺CCR7⁺ T_CM and CD4⁺CD45RO⁺CCR7⁻ T_E/EM cells. Numbers indicate the percentage of cells in each gate. (B) Detection of IL-2, TNF-α and IFN-γ production by CD4⁺ T_CM and CD4⁺T_E/EM cells. Numbers indicate the percentage of cytokine⁺ cells for each memory cell type. (C) Boolean gating analysis was used to divide cytokine-producing cells into seven distinct populations based on their production of IL-2 (2), TNF-α (T) and IFN-γ (G) in any combination. Each population is shown as two dot plots: the upper is TNF-α vs IL-2 and the lower is IFN-γ vs. IL-2. Numbers indicate the percentage of cytokine⁺ cells for each memory subtype.</p

Frequency of CD4^<b>+</b> T_CM and CD4^<b>+</b> T_E/EM cells producing IL-2 upon recall with CSP peptides.

<p>PBMC were obtained at the Pre, Po2, DOC and PoCh timepoints from RTS,S-immunized subjects and were incubated for 18 h with co-stimulants (medium control) or co-stimulants plus a pool of 2 long contiguous CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cultures also contained PE-conjugated-CCR7-specific Ab to detect CCR7⁺ cells. Cells were harvested and surface-labeled for CD3, CD4, CD8 and CD45RO and then stained for intracellular detection of IL-2, TNF-α and IFN-γ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>). Cells were analyzed on an LSR-II flow cytometer to identify IL-2-producing CCR7⁺CD45RO⁺CD4⁺ T_CM cells and CCR7⁻CD45RO⁺CD4⁺ T_E/EM cells. The frequency of IL-2-producing CD4⁺ T cell subsets in the medium control cultures were subtracted from the corresponding values for the CD4⁺ T cell subsets in the cultures containing Ag. The resulting adjusted values are plotted as [A] the % IL-2 producing CD4⁺ T_CM cells and [B] the % IL-2 producing CD4⁺ T_E/EM cells for protected (closed symbol) and non-protected (open symbol) subjects. Statistically significant differences between pre-immune and post-vaccination time points are indicated at the top of the graph. Statistically significant differences between values for protected and non-protected subjects are as indicated within the body of the graph. *p<0.05; **p<0.01; ***p<0.001.</p