1. Control of Plasmodium falciparum erythrocytic cycle: γδ T cells target the red blood cell–invasive merozoites
by Giulia Costa, Séverine Loizon, Marianne Guenot, Iulia Mocan, Franck Halary, Geneviève de Saint-Basile, Vincent Pitard, Julie Déchanet-Merville, Jean-François Moreau, Marita Troye-Blomberg, Odile Mercereau-Puijalon, and Charlotte Behr
Blood
Volume 118(26):
December 22, 2011
©2011 by American Society of Hematology

2. Antiplasmodial activity of Vγ9Vδ2 T cells in PBMCs and as purified T-cell lines.
Antiplasmodial activity of Vγ9Vδ2 T cells in PBMCs and as purified T-cell lines. (A) Undepleted PBMCs (total PBMCs) or Vδ2+ T cell–depleted PBMCs (Vδ2-depleted PBMCs) from 8 different healthy donors were activated with BrHPP (400nM) or left untreated for 40 hours and then cocultured in a standard parasite inhibition assay at a 4/1 E/T ratio with a synchronized trophozoite culture for 24 hours. At the end of this period, parasitemia in cocultured samples was compared with that in synchronized trophozoite cultures incubated without PBMCs by hydroethidine staining. The percentage of Vδ2+ CD3+ cells among total PBMCs varied among the donors (from 0.5% to 1.9%). Data represent the parasitemia (means of duplicates) of cocultures with PBMCs of each donor tested (n = 8; *P ≤ .05 by the Wilcoxon signed ranked test). (B) Short-term Vγ9Vδ2 T-cell lines (STL) were tested after a 24-hour priming with no cytokine added or with IL-2 (20 IU/mL) only or with IL-15 (50 ng/mL) and cocultured with the parasites at a 4/1 E/T ratio in a standard parasite inhibition assay. Data represent the mean parasitemia in the various cocultures conditions (n = 10 STL; **P ≤ .01 for Wilcoxon signed ranked test comparisons). (C) S1 and the JT long-term Vγ9Vδ2 T-cell lines were (i) primed for 24 hours with IL-2 (20 IU/mL) and increasing doses of IL-15 and cocultured with the parasites at a 4/1 E/T ratio or (ii) primed for 24 hours with IL-2 (20 IU/mL) with or without added IL-15 (50 ng/mL) and cocultured with the parasites at increasing E/T ratios as indicated. Graphs represent the mean of parasitemia ± SD of 4 independent experiments (n = 4; *P ≤ .05 and **P ≤ .01 by Mann-Whitney rank sum test; ns indicates not significant).
Giulia Costa et al. Blood 2011;118:
©2011 by American Society of Hematology

3. Perforin is dispensable for Vγ9Vδ2 antiparasitic activity.
Perforin is dispensable for Vγ9Vδ2 antiparasitic activity. A Vγ9Vδ2 T-cell line was generated from a perforin-deficient familial hemophagocytic lymphohistiocytosis (FLH) patient, and its antiparasitic activity was assessed in a standard parasite inhibition assay. (A) Flow cytometry histograms showing perforin and granulysin expression in Vγ9Vδ2 T-cell lines generated from 2 different donors (S1 and JT) and from an FLH patient (PFNdef). Mean fluorescence intensity (MFI) for perforin and granulysin intracellular staining is indicated in the top right corner of each panel. Dotted lines represent isotype control antibody. (B) Comparison of the antiparasitic activity of the PFNdef, S1 and JT cell lines. The αβ T4A.5 T-cell clone was used as a negative control. Antiparasitic activity (percentage) was calculated as 100 − [(average % parasitemia of duplicate with cells in the presence of IL-2 + IL-15/average % parasitemia of duplicate with cells in the presence of IL-2) × 100]. Data represent the mean ± SD of 4 independent experiments (n = 4; *P ≤ .05 by Mann-Whitney rank sum test comparing the PFNdef cell line with the S1, JT, or T4A.5 T-cell lines).
Giulia Costa et al. Blood 2011;118:
©2011 by American Society of Hematology

4. Granulysin is essential for Vγ9Vδ2 antiparasitic activity.
Granulysin is essential for Vγ9Vδ2 antiparasitic activity. The S1 T-cell line was infected with the lentiviral vector containing the granulysin-specific shRNA sequences GNLYsh1, GNLYsh2, or a NS shRNA sequence, and their antiparasitic activity has been assessed in a standard parasite inhibition assay. (A) RT-PCR assessment of granulysin mRNA levels in the noninfected S1 cell line (Ctrl) or S1 cell line infected with NS, GNLYsh1 (Gnly1), or GNLYsh2 (Gnly2) shRNA constructs. β2-Microglobulin mRNA levels were measured as a control. (B) Granulysin and perforin protein levels were detected in S1 (Ctrl), NS, Gnly1, and Gnly2 cell lines by Western blotting. Actin expression was measured as a loading control. (C) Intracellular granulysin expression was measured using flow cytometry in the NS, Gnly1, and Gnly2 cell lines. The percentage of granulysin-positive cells and the MFI of granulysin intracellular staining are indicated in the top right corner of each panel. Dotted lines represent isotype control antibody. (D) Antiparasitic activity of the NS, Gnly1, and Gnly2 cell lines. Antiparasitic activity (percentage) was calculated as 100 − [(average % parasitemia of duplicate with cells in the presence of IL-2 + IL-15/average % parasitemia of duplicate with cells in the presence of IL-2) × 100]. Data represent the mean ± SD of 2 independent experiments (n = 2; *P ≤ .05 by Mann-Whitney rank sum test comparing NS with Gnly1 and Gnly2 T-cell lines).
Giulia Costa et al. Blood 2011;118:
©2011 by American Society of Hematology

5. Vγ9Vδ2 T cells degranulate in the presence of trophozoites and merozoites in a TCR-dependent mechanism.
Vγ9Vδ2 T cells degranulate in the presence of trophozoites and merozoites in a TCR-dependent mechanism. The S1 and JT Vγ9Vδ2 T-cell lines were incubated for 6 hours with uiRBCs, purified schizonts (Schiz), or merozoites (Mero), and CD107a surface expression was detected as described in “Methods.” (A) Representative flow cytometry dot-plot showing the gating strategy for Vδ2+ T cells and for detection of CD107a surface expression on gated cells after incubation with uiRBCs or schizonts. (B) Percentage of CD107a+ cells from S1 and JT cell lines incubated in CM or with uiRBCs, purified schizonts, or merozoites at decreasing E/T ratios (1/10, 1/5, and 1/2.5). Data represent mean ± SD of 4 independent experiments (n = 4) and were obtained by collecting of Vδ2+ events (*P ≤ .05 by Mann-Whitney rank sum test comparing cells incubated with uiRBCs, schizonts, or merozoites with cells incubated in CM). (C) Percentage of CD107a+ cells among S1 and JT cells, or among control T cells (a Vγ4Vδ5 T-cell line, a Vδ3VγI T-cell line, and an αβ T4A.5 T-cell clone) incubated in uiRBCs, purified schizonts, or merozoites at 1/10 E/T ratio. The figure is representative of 3 independent experiments (n = 3). (D) Percentage of CD107a+ cells among S1 (top) and JT cells (bottom) preincubated for 1 hour with 1, 0.5, 0.1, and 0.01 μg/mL of anti-Vδ2 (IgG1, clone immu389) or anti-NKG2D (IgG1, clone ) blocking antibodies and then incubated with uiRBCs, purified schizonts, or merozoites at 1/10 E/T ratio. Data represent mean ± SD of 4 independent experiments (n = 4) and were obtained by collecting Vδ2+ events (*P ≤ .05 by Mann-Whitney rank sum test comparing cells incubated with different concentrations of anti-Vδ2 or anti-NKG2D blocking antibodies with untreated cells on schizont or merozoite stimulation).
Giulia Costa et al. Blood 2011;118:
©2011 by American Society of Hematology

6. Merozoites, not trophozoites, are the target of Vγ9Vδ2 antiparasitic activity.
Merozoites, not trophozoites, are the target of Vγ9Vδ2 antiparasitic activity. (A) Primed S1 or JT Vγ9Vδ2 T cells were cocultured with purified trophozoites (E/T ratio 4/1) for either 24 hours until merozoite reinvasion (no removal of γδ) or were removed by magnetic depletion after 6 hours of coculture, just before schizont rupture; [removal of γδ(T6h)]. Mock-treated parasites cultured without cells were used as a control. After cell removal, the parasitemia was similar under the various conditions. (i) Schematic representation of the experimental design. (ii) Antiparasitic activity was calculated as 100 − [(average % parasitemia of duplicate with cells in the presence of IL-2 + IL-15/average % parasitemia of duplicate with cells in the presence of IL-2) × 100]. Data represents the mean ± SD of the antiparasitic activity observed in 3 independent experiments performed in duplicate (*P ≤ .05 for Mann-Whitney rank sum test comparing the conditions [removal of γδ] and [no removal of γδ] for each T-cell line). (iii) Histograms represent the mean of parasitemia ± SD observed in 1 representative experiment performed in duplicate with the S1 and the JT cell lines. No cells: parasites cultured in the absence of cells (*P ≤ .05 by Mann-Whitney rank sum test comparing the parasitemia of parasites cultured in the presence of T cells [S1 or JT] with the parasitemia in absence of T cells [no cells], for each condition (that is, [removal of γδ] or [no removal of γδ]). Flow cytometry histograms show granulysin expression in the S1 and JT cell lines before (T0) and after 6 hours (T6hr) of coculture with trophozoite-stage parasites. Dotted lines represent isotype control antibody. (B) Purified merozoites were incubated with fresh uiRBCs alone (no cells) or in the presence of S1 or JT T cells or the αβ T4A.5 T-cell clone, and the parasitemia was assessed after 28 hours. (i) Schematic representation of the experimental design. (ii) Inhibition of merozoite reinvasion (percentage) was calculated as 100 − [(average % parasitemia with cells/average % parasitemia without cells) × 100]. Data represent the mean ± SD of the inhibition of merozoite invasion observed in 3 independent experiments performed in duplicate (n = 3; *P ≤ .05 by Mann-Whitney rank sum test comparing S1 or JT with T4A.5). (iii) Parasitemia from 1 representative experiment is shown (mean ± SD; *P ≤ .05 by Mann-Whitney rank sum test comparing the parasitemia in the presence of T cells [S1, JT, or T4A.5] with the parasitemia in absence of T cells [no cells]).
Giulia Costa et al. Blood 2011;118:
©2011 by American Society of Hematology

7. Granulysin and Vδ2+ T-cell phenotype in primary-infected patients.
Granulysin and Vδ2+ T-cell phenotype in primary-infected patients. (A) PBMCs from P falciparum primary-infected patients and healthy donors were stained ex vivo for intracellular granulysin and effector/memory surface markers. (i) Representative flow cytometry dot-plot outlining the gating strategy for Vδ2+ T cells (CD3+Vδ2+ lymphocytes). Flow cytometry histograms showing granulysin expression in Vδ2+ gated T cells (black line) compared with isotype control antibody (dotted line) in a representative patient and healthy donor (control). The percentage of positive cells for granulysin intracellular staining is indicated in the top right corner of each panel. (ii) Percentages of CD3+Vδ2+ cells positive for granulysin in malaria patients and healthy donors (controls). The box represents the 75th and 25th percentiles, and the bar represents the median. P value was determined using Mann-Whitney rank sum test to compare patients (n = 7) with controls (n = 10). Data were obtained by collecting total events. (Bi) Representative example of flow cytometry data showing the gating strategy for Vδ2+ T cells (CD3+Vδ2+ lymphocytes). Expression of the CD27 and CD45RA cell surface markers in the gated Vδ2+ T cells define distinct effector/memory subpopulations (TNAIVE, TCM, TEM, and TEMRA). (ii) Data represent the mean ± SD of the percentage of Vδ2+ T cells of each effector/memory subset from patients or controls (*P ≤ .05 by Mann-Whitney rank sum test comparing patients [n = 6] with controls [n = 10]). Data were obtained by collecting total events. (Ci) Plasma granulysin levels in patients and controls (i) were detected by ELISA. Box represents the 75th and 25th percentiles, and the bar represents the median. The P value was determined using Mann-Whitney rank sum test to compare patients (n = 12) with controls (n = 13). (ii) Log-transformed levels of plasma granulysin plotted against the percentage of CD3+ T cells expressing Vδ2 in patients. Statistical analysis was performed using Spearman rank correlation test, and the ρ and the P value are indicated (n = 7). (D) Fresh PBMCs from 2 patients (P1 and P2) were cocultured with purified trophozoites (iRBCs) or uiRBCs at a 1/5 E/T ratio. CD107a surface expression detected after 6 hours of coculture was measured. The percentage of CD3+Vδ2+ T cells or CD3+Vδ2− T cells expressing CD107a is shown. Data were obtained by collecting total events.
Giulia Costa et al. Blood 2011;118:
©2011 by American Society of Hematology