Flow-cytometry assessment of parasitemia in cell parasite co-culture assay using hydroethidine staining Western blotting RT-PCR

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1 Flow-cytometry assessment of parasitemia in cell parasite co-culture assay using hydroethidine staining The capability of cells, either PBMC or purified T- cell lines, to inhibit parasite growth or merozoite invasion, was assessed by flow cytometry on duplicate wells using hydroethidine (HE) staining as previously described (Van der Heyde HC et al, Clin.Diagn.Lab.Immunol., 1995, 2: ). HE is a membrane permeant DNA probe that needs to be dehydrogenated to intercalate into DNA and thus stains only live parasites or cells. Co-cultures were incubated with HE (Polyscience, Warrington, USA) at the final concentration of 5 µg/ml during 20 minutes at 37 C, then washed twice in PBS and re-suspended in 3 ml of PBS before analysis by FACSCanto (BD Bioscience, USA). A minimum of 1000 events was acquired in the region corresponding to infected erythrocytes i.e. HE positive erythrocytes. Western blotting Vγ9Vδ2 T cell lines were lysed in buffer containing 50 mm Tris-HCl, ph 7.4, 150 mm NaCl, 1% Triton X-100, supplemented with phosphatase and protease inhibitors. Protein extracts were quantified using a BCA Protein Assay kit (Thermo Scientific, USA). Aliquots of total extracts were separated by electrophoresis on 12% SDS-PAGE gels under reducing conditions. Membranes were blotted with mouse anti-human granulysin Ab (clone RF10, MBL), mouse anti-human perforin Ab (clone ebiobor21, ebioscience Inc.) and mouse antihuman actin Ab (Sigma Aldrich). RT-PCR Total RNA was extracted from Vγ9Vδ2 T cell lines using TRIzol reagent (Invitrogen) according to the manufacturer s recommendations. From each sample, 1.5 µg total RNA was reverse transcribed using M-MLV Retrotranscriptase (Invitrogen) and amplified using PCR reagents (Promega, USA) according to the manufacturer s protocol. The primers used were as follows: granulysin, forward 5 -GGCCGTGACTACAGGACCTGTC-3 and reverse 5 - CCTGAGGTCCTCACAGATCTG-3 19 ; β2 -microglobulin, forward 5 - AATTGAAAAAGTGGAGCATTCAGA-3 and reverse 5 - GGCTGTGACAAAGTCACATGGTT-3.

2 Granulysin ELISA Briefly, Maxisorp plates (Nunc) coated with 4 µg/ml RB1 mab (MBL) were blocked with PBS/0.1% Tween-20/2% BSA and serially incubated with samples or standards (R&D Systems), 1 µg/ml biotinylated RC8 mab (MBL), HRP-conjugated streptavidin (GE Healthcare) and the substrate Tetraethyl Benzedrine (R&D) with washing steps between each incubation.

3 Table S1. Functional characterization of Vγ9Vδ2 short-term T-cell lines Short-term Vγ9Vδ2 T cell lines were generated from PBMCs from healthy donors (n=14) after phosphoantigen stimulation as described in Materials and Methods. Between 11 and 18 days after generation, cell lines were tested for expression of the Vδ2 chain and granulysin, and degranulation activity was assessed in Vδ2 + cells upon stimulation with purified schizonts (Schiz) and merozoites (Mero) using a CD107a assay. When the proportion of CD3 + T cells expressing Vδ2 reached at least 75% (STLA to STLJ), cell lines were also tested for their capacity to inhibit parasite growth and merozoite invasion. All tests were performed as described in Materials and Methods. Antiparasitic activity was calculated as follows: [(average % parasitemia of duplicate with cells in the presence of IL-2+IL15/ average % parasitemia of duplicate in the absence of cells)x100]. ND: Not Determined.

4 Figure S1. Gating strategy for assessment of parasitemia in cell parasite co-culture assay using HE staining Parasites and cells from a co-culture were stained by HE as described in supplemental methods. Cells debris were excluded from the gate P1 based on FSC/SSC. The percentage of parasitemia was calculated in the P2 gate corresponding to total erythrocytes as followed: [number of infected erythrocytes (P3) / number of total erythrocytes (P2)] X 100 using the FACSDiva program (BD Biosciences). The total erythrocytes gate (P2) excludes cells (i.e. leukocytes) (P4), which are one-log more fluorescent than infected erythrocytes according to their higher DNA content. Representative flow-cytometry dot-plots and histograms outlining the gating strategy are shown: Upper panel uninfected erythrocytes (uirbcs) alone; middle panel, parasites culture without cells (irbcs: infected erythrocytes); lower panel: cellsparasites co-culture.

5 Isotype CD56 CD94 NKG2A NKG2C NKG2D CD85j S1 13 2, , ,659 1,526 1, , JT S1 CD158a CD158b CD158e 17 CD158i NKp3 NKp NKp JT Figure S2. Phenotypic characterization of NK receptors in Vγ9Vδ2 long-term T cell lines The S1 and JT Vγ9Vδ2 T cell lines were restimulated as described in Materials and Methods, stained for different NK receptors and then analyzed using flow cytometry. Briefly, γδ T cells were incubated with isotype control antibodies or mabs directed against the following cell surface markers: CD56, CD94, NKG2A, NKG2D, CD158a, CD158b, CD158e, CD158i, NKp30, NKp44, and CD85j (BD Biosciences), and NKp80 and NKG2C (R&D Systems). The mean fluorescence intensity (MFI) of each surface marker is indicated in the upper right corner of each panel.

6 Lower chamber parasitemia (%) RPMI P RPMI γδ+p γδ P γδ+p P S1 J Figure S3. Inhibition of parasite growth is contact-dependent To determine whether contact is required to inhibit parasite growth, experiments were performed (E/T ratio = 4/1) using Transwell inserts to physically separate the γδ T cells (γδ) from the parasites (P). Parasitemia was assessed in the lower chamber using hydroethidine staining after 24 hours. In control samples, the upper chamber contained RPMI 1640 medium, and the lower chamber contained the parasite culture alone or the parasite-γδ T cell coculture.

7 Hydroethidine Isotype Fas DR4 DR5 Figure S4. Neither infected nor uninfected red blood cells express death receptors Parasite cultures (10% parasitemia, schizont stage) were stained with isotype control IgG or purified anti-fas (clone 5D7, a kind gift of Dr. P. Legembre), purified anti-dr4, or purified anti-dr5 (Alexis) mab for 30 min at 4 C. Cells were washed twice and stained with goat anti-mouse Abs (Invitrogen) according to the manufacturer s recommendations. Parasites were then stained with hydroethidine to discriminate infected from uninfected red blood cells as described in Materials and Methods. Surface marker expression was assessed using flow cytometry. As positive controls, the Jurkat and the SKM 6.4 cell lines were stained under the same conditions; these cells displayed positive staining (data not shown). Dot plots depict hydroethidine levels versus death receptor expression levels. Note that the infected erythrocytes were positive for hydroethidine, whereas the uninfected erythrocytes were negative.

8 TNFα production (pg/ml) Figure S5. Granulysin-silenced cell lines produce TNF-α at levels similar to the nonsilenced control cell line To control for possible functional disturbances inherent to granulysin silencing, granulysinsilenced and non-silenced Vγ9Vδ2 S1 T cells were tested for TNF-α production. Noninfected S1 T cells (Ctrl) and S1 T cells infected with lentivirus expressed non-silencing shrna (NS) or granulysin-specific shrnas (Gnly1 and Gnly2) were incubated for 6 hours with BrHPP (200 nm) or complete medium alone (CM), and TNF-α release was quantified by ELISA (Bender MedSystems). The results are presented as the mean ± SD of two independent experiments performed in duplicate (n=2). There was no significant difference (ns) between the Gnly1 and Gnly2 cell lines and the NS control cell line (p > 0.05 by the Kruskal-Wallis test).

9 Figure S6. The anti-parasitic activity of short-term γδ T cell lines correlates with the granulysin concentration in the supernatants Short-term Vγ9Vδ2 T cell lines (n=10; STLA to STLJ) were tested in a standard parasite inhibition assay as described in Materials and Methods (Supplemental Table 1). At the end of the inhibition tests, supernatants were collected, and granulysin concentrations were assessed by ELISA (see Supplemental Methods). The anti-parasitic activity of each of the Vγ9Vδ2 T cell lines was tested and plotted against the granulysin concentration in the supernatant. Statistical analysis was performed using the Spearman's rank correlation test, and rho and p values are indicated on the graph (n=10).

10 Figure S7. Reinvasion and development of purified merozoites Purified merozoites were obtained as described in Materials and Methods. (Left) Purified merozoite clusters were observed in methanol-fixed Giemsa-stained thin smears. Free merozoites were subsequently co-cultured with uninfected red blood cells, and parasite development was monitored by Giemsa staining after 7 hours (middle) and 28 hours (right). Representative images from one experiment is shown.