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Clinical Trial
. 2012 May 15;188(10):5054-62.
doi: 10.4049/jimmunol.1102710. Epub 2012 Apr 13.

Antigen-specific IL-2 secretion correlates with NK cell responses after immunization of Tanzanian children with the RTS,S/AS01 malaria vaccine

Amir Horowitz  1 , Julius C R Hafalla, Elizabeth King, John Lusingu, Denise Dekker, Amanda Leach, Philippe Moris, Joe Cohen, Johan Vekemans, Tonya Villafana, Patrick H Corran, Philip Bejon, Chris J Drakeley, Lorenz von Seidlein, Eleanor M Riley
Affiliations
Clinical Trial

Antigen-specific IL-2 secretion correlates with NK cell responses after immunization of Tanzanian children with the RTS,S/AS01 malaria vaccine

Amir Horowitz et al. J Immunol. .

Abstract

RTS,S/AS01, a vaccine _targeting pre-erythrocytic stages of Plasmodium falciparum, is undergoing clinical trials. We report an analysis of cellular immune response to component Ags of RTS,S-hepatitis B surface Ag (HBs) and P. falciparum circumsporozoite (CS) protein-among Tanzanian children in a phase IIb RTS,S/AS01(E) trial. RTS,S/AS01 (E) vaccinees make stronger T cell IFN-γ, CD69, and CD25 responses to HBs peptides than do controls, indicating that RTS,S boosts pre-existing HBs responses. T cell CD69 and CD25 responses to CS and CS-specific secreted IL-2 were augmented by RTS,S vaccination. Importantly, more than 50% of peptide-induced IFN-γ(+) lymphocytes were NK cells, and the magnitude of the NK cell CD69 response to HBs peptides correlated with secreted IL-2 concentration. CD69 and CD25 expression and IL-2 secretion may represent sensitive markers of RTS,S-induced, CS-specific T cells. The potential for T cell-derived IL-2 to augment NK cell activation in RTS,S-vaccinated individuals, and the relevance of this for protection, needs to be explored further.

Trial registration: ClinicalTrials.gov NCT00380393.

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Figures

Figure 1
Figure 1. T cell responses to HBs and CS peptides in vaccinees and controls
PBMCs were cultured in growth medium alone (GM), with HBs or CS peptides or with rIL-12/IL-18 for 24 h and analysed by flow cytometry. (A) An example of the gating strategy for CD3+ and CD4+ T cells, CD69, IFN-γ and CD25 expression for one RTS,S/AS01E vaccinated subject. (B) Percentage of T cells expressing IFN-γ (left plots) and mean MFI for CD69 (middle plots) and CD25 (right plots) expression on T cells in RTS,S/AS01E vaccinees (upper plots) and controls (lower plots). Fold change in T cell IFN-γ % (C), T cell CD69 MFI (D) and CD4+ T cell CD25 MFI (E). The horizontal line within each box represents the median; the top and bottom of each box represent the 25th and 75th percentiles, respectively, and the whiskers represent the upper and lower limits of values within 1.5 times the interquartile range. Circles denote outliers. Data from 80 RTS,S/AS01E vaccinees and 98 controls is presented for IFN-γ and CD69; data from 55 RTS,S/AS01E vaccinees and 61 controls is presented for CD25. P values are derived from paired Wilcoxon (B) and unpaired Mann Whitney (C,D,E) tests.
Figure 2
Figure 2. Cytokine secretion from HBs and CS peptide stimulated PBMCs in vaccinees and controls
PBMCs were cultured in growth medium alone (GM), with HBs or CS peptides for 24 h and culture supernatants from 38 RTS,S/AS01E vaccinees and 54 controls were collected and analyzed by Luminex bead array for (A) IFN-γ, (B) IL-2 and (C) IL-10. The horizontal line within each box represents the median; the top and bottom of each box represent the 25th and 75th percentiles, respectively, and the whiskers represent the upper and lower limits of values within 1.5 times the interquartile range. Circles denote outliers. P values are derived from paired Mann Whitney tests.
Figure 3
Figure 3. NK cell responses among HBs and CS peptide stimulated PBMCs in vaccinees and controls
PBMCs from RTS,S/AS01E vaccinees (n = 80) and controls (n = 98) were cultured in growth medium alone (GM), with HBs or CS peptides or with rIL-12/IL-18 for 24 h and analysed by flow cytometry. (A) An example of the gating strategy for NK cells (CD3-CD56+), CD69 and IFN-γ expression for one RTS,S/AS01E vaccinated subject. (B) Percentage of NK cells expressing IFN-γ (left plots) and mean MFI for CD69 expression on NK cells (right plots) in RTS,S/AS01E vaccinees (upper plots) and controls (lower plots). Fold change in NK cell IFN-γ % (C) and NK cell CD69 MFI (D). The horizontal line within each box represents the median; the top and bottom of each box represent the 25th and 75th percentiles, respectively, and the whiskers represent the upper and lower limits of values within 1.5 times the interquartile range. Circles denote outliers. P values are derived from paired Wilcoxon (B) and unpaired Mann Whitney (C,D) tests.
Figure 4
Figure 4. Correlation between IL-2 secretion and NK cell IFN-γ or CD69 responses to HBs and CS peptides
NK cell IFN-γ % (A,C,E,G) and CD69 MFI (B,D,F,H) responses were compared with IL-2 secretion (pg/ml) for PBMC cultured with (A-D) HBs or (E-H) CS peptides for 24 h, from RTS,S/AS01E vaccinees (n = 39, A,B,E,F) and controls (n = 55, C,D,G,H). Each circle represents data for one individual child. Solid lines = the goodness of fit (r2) as determined by Spearman’s correlation analysis; dotted lines = 95% confidence band.
Figure 5
Figure 5. Relative contribution of T cells and NK cells to the total IFN-γ response to HBs and CS peptides in RTS,S/AS01E vaccinated subjects
PBMCs from RTS,S/AS01E vaccinees (n = 80) were cultured with HBs or CS peptides for 24 h and analysed by flow cytometry. (A) An example of the gating strategy to identify IFN-γ+ cells and then to determine the proportion of these cells that are either T cells (CD3+), NK cells (CD3−, CD56+) or CD3−CD56− cells for HBs-stimulated cells from one vaccinated subject. (B) Bar charts indicate the absolute number (mean/SD per 105 PBMCs) of IFN-γ+ cells that are NK cells, T cells or CD3−CD56− (DN, double negative) lymphocytes among HBs-stimulated (filled bars) or CS-stimulated (open bars) PBMC.
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