Eryptosis of iRBC after co-culture with PBMCs or purified NK cells.

<p>iRBC were co-cultured 3∶1 for 24 hours with NK cells, NK cells+TKD (5 day stimulation), PBMC, PBMC+TKD (5 day stimulation) of the same donor. 0.5×10⁶ erythrocytes were washed in 5 mM Ringer solution. Afterwards, cells were stained for 15 minutes with Annexin-V (1∶500) and propidium iodide (1∶50). Eryptotic cells were determined as Annexin-V-positive (AV⁺) and propidium iodide-negative (PI⁻). A: Baseline levels of eryptotic uRBC and iRBC at the start of the experiment and after 24 h of culture in growth medium without leukocytes. RBC were stained with AV+PI or left unstained. Gating was done based on FSC/SSC properties and the unstained control. AV was measured in the FL1 channel and PI in the FL2 channel. B: Percentage of AV⁺/PI⁻-iRBC after co-culture in growth medium (GM) or with different effector cells in relation to starting parasitemia (** p≤0.01, student's <i>t</i> test, n = 6). C: Normalized FSC of iRBC after co-culture in growth medium (GM) or with different effector cells (* p≤0.05, student's <i>t</i> test, n = 6). FSC values of co-cultured iRBC were normalized to untreated iRBC (GM). D: PI⁺ necrotic iRBC after culture in growth medium (GM) or with various effector cells. Numbers of necrotic cells were normalized to untreated iRBC (GM).</p

Growth delay in <i>Plasmodium falciparum</i> development after NK cell contact.

<p>A: Representative figure of 3D7-iRBC before the start of co-cultures. Parasites were synchronized by magnetic cell sorting columns for late stages. B: Representative figure of 3D7-iRBC after 24 h of incubation. The parasites have developed into normal ring-stage forms that are expected for this time point. Infected RBC were either co-cultured 3∶1 with autologous NK cells (C), NK cells+TKD (pre-stimulated for 5 days before co-culture with 2 µg/ml TKD peptide) (D), PBMCs (E), or PBMCs stimulated with TKD peptide (F). Normal parasite development of a control culture of parasites without leukocytes was observed in parallel. A blood smear was prepared before and after 24 hours of incubation and stained with 10% Giemsa. Experiments were repeated with RBC from 3 different donors.</p

Flow cytometry analysis of erythrocytes for possible NK cell ligands.

<p>0.5×10⁶ i/uRBC or Hela cells were stained with anti-hMICA/B-PE (A), anti-hHLA-E-PE (B) or the respective isotype control. As a control, 0.5×10⁶ iRBC, uRBC or NK92 cells were also stained with anti-hHLA-E-PE (B) or a PE-isotype control. The presence of Hsp70 on uRBC (C) and iRBC (D, E) was determined with anti-Hsp70 mAb cmHsp70.1-FITC compared to FITC isotype. Parasite DNA was stained with Hoechst dye (schizonts, D) that is detected in the Pacific blue channel or Hydroethidine (rings, E) that is detected in the PE channel. Experiments were repeated 3 times.</p

Transcriptional and translational changes of GzmA, GzmB and Perforin in NK92 cells after 24 hours stimulation.

<p>NK92 cells were left untreated (ns) or cultured with IL-12/-18 (IL), IFN-α, uRBC or iRBC (1∶3) for 24 h. A: Changes on transcriptional level of stimulated cells in comparison to untreated cells were analyzed for GzmA, GzmB and Perforin. β-Actin expression served as house-keeping gene normalizer. Data are represented as mean ± SD (student's <i>t</i> test * p<0.05; ** p<0.01) and are representative of three independent experiments each performed in duplicate. B: After 24 h, 1×10⁷ NK92 cells were lysed, incubated 30 min on ice and subsequently centrifuged at 13000× <i>g</i> for 15 min at 4°C. 10 µg of total supernatant protein were separated by 7.5% SDS-PAGE and blotted onto a nitrocellulose membrane. After blocking, membranes were incubated for 1 h with anti-β-Actin (lane 1), anti-hGzmA (lane 2), anti-hGzmB (lane 3), or anti-hPrf (lane 4).</p

Granzyme B-Elispot of NK92 cells (A) or isolated NK cells (B,C) after 24 hours of co-culture with i/uRBC.

<p>Some cultures were pre-activated 5 days with TKD peptide or the scrambled NGL peptide and/or pre-incubated with blocking Hsp70 antibody (cmHsp70.2) or a blocking antibody IgM-isotype control for 20 minutes before the start of the experiment. After stimulation, 2000 NK cells were cultured either alone, with iRBC or uRBC (1∶3 or 10∶1) on a GzmB antibody-coated 96-well plate. GzmB-releasing cells were counted after 24 hours of incubation (* p≤0.05, ** p≤0.01, *** p≤0.001, student's <i>t</i> test, n = 6).</p

Hypothetical model of NK cell response to iRBC and senescent uRBC.

<p>If erythrocytes become senescent (A) or are infected with <i>Plasmodium falciparum</i> (B,C) host-Hsp70 will be recruited to the cell membrane. ?1 NK cells recognize Hsp70-exposure by yet unknown receptors, possibly CD94. Recognition of Hsp70 leads to GzmB release. ?2 GzmB will enter the target cell with either assistance of Hsp70, an unknown receptor or become endocytosed. ?3 Once inside the iRBC, GzmB induces eryptosis. Pre-stimulation with TKD peptide enhances both GzmB release and the amount of eryptotic iRBC (C).</p

Antibody titers against the three vaccine strains at baseline (day 0), day 28 and day 84.

<p>Red lines indicate the mean of all volunteers of the antihelminthic treated group (AT) and blue lines indicate the mean of all participants of the placebo group. Dashed lines indicate antibody titers of each participant.</p

Differences of HI titers between the respective visits (day 28, day 84) and day 0 (baseline).

<p>Red and blue colors represent the pre-treated (AT) and control group.</p

Vaccine specific IgA at day 0, day 28 and day 84 in antihelminthic treated (AT) (red) and placebo group (blue).

<p>Vaccine specific IgA at day 0, day 28 and day 84 in antihelminthic treated (AT) (red) and placebo group (blue).</p

<i>Var</i> transcription profiles of parasites recovered from infected volunteers at the day of first microscopically detectable parasitemia.

<p>(A) Heat map showing the individual <i>var</i> gene expression for all volunteer samples taken when parasites were present in the thick blood smear ranked by mean expression. To correct for individual differences in the overall <i>var</i> expression levels, the expression for each <i>var</i> gene was normalized against the total <i>var</i> expression in each sample. The color scale indicates the relative expression levels with red representing values above the median, blue representing values below the median, and white representing median. Grey means not detected. The number of sporozoites (200, 800, 2500 and 3200) and mode of injection (iv = intravenous, id = intradermal) used for each volunteer are indicated below. (B) The distribution of the relative gene expression per <i>var</i> gene and control genes is shown in a dot plot for all volunteer samples at the day of patent infection defined as parasites present in the thick blood smear (n = 18). Each point represents a <i>var</i> gene expression value relative to the normalizing gene <i>sbp1</i> observed per volunteer sample and the median expression per <i>var</i> gene is marked. Housekeeping genes used as controls, <i>var</i> gene names and groups are indicated. (C) Proportion of <i>var</i> gene expression by group across all volunteers at the day of patent infection. For comparison, genomic proportion of each <i>var</i> gene group is indicated after the color code. (D) Comparison of the expression levels between subtelomeric and centromeric <i>var</i> gene variants. The box plot shows the distribution of transcript levels for each individual <i>var</i> gene relative to <i>sbp1</i> according to the chromosomal localization of the genes for all 18 volunteer samples at the day of patent infection. Gene expression varied significantly between both gene sets (Wilcoxon rank-sum test, p<0.0001) with median expression of 232 (IQR: 87–510) for telomeric <i>var</i> genes and 40 (IQR: 18–94) for centromeric <i>var</i> genes. (E) The heat map of pairwise Spearman’s rank correlation coefficients (R) between expression profiles illustrates the positive correlation between all 18 volunteer samples at the day of patent infection. Volunteer samples were ranked by the sum of their correlation coefficients. The color scale indicates the correlation coefficient in the range from 0 to 1. With exception of isolate 25.1 versus the isolates 02.1 (p = 0.0015), 25.3 (p = 0.0029) and 32.4 (p = 0.0030) all expressions correlated at a significance level below 0.001.</p