γδ T cells suppress Plasmodium falciparum blood-stage infection by direct killing and phagocytosis

Abstract

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum-infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria-γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs.

Extended Data 1. Gating strategy for phenotypic analysis of peripheral blood lymphocytes from healthy donors (HD) and P. falciparum-infected patients (Pf)

a, PBMCs were stained with the Live/Dead viability dye and antibodies to CD3, CD4, CD8, TCRδ2 and CD56. Single live cells were gated on SSC-A vs FSC-A and dead cells were excluded. Lymphocyte subpopulations were gated as: CD3⁺CD4⁺ (CD4⁺ T cells), CD3⁺CD8⁺ (CD8⁺ T cells), CD3⁺TCRδ2⁺ (γδ2 T cells) and CD3⁻CD56⁺ (NK). b, PBMCs co-stained for CD69, CD16 and cytotoxic effector protein expression (GNLY, PFN and GzmB) were gated on γδ2 T cells.

Extended Data 2. Gating strategy to measure RBC lysis

a, γδ2 T cells stained with anti-γδ2-PE were purified by positive selection with anti-PE microbeads and cell purity was evaluated by flow cytometry co-staining with anti-CD3. b, Infected or uninfected RBCs were stained with CFSE prior to co-culture with γδ2 T cells. After co-culture, cells were stained with anti-CD3 (γδ2 T cells) and anti-CD235a (RBCs). An equivalent number of counting beads as CSFE-stained RBCs (before γδ2 T cell coculture) was added to each condition prior to flow cytometry acquisition. A CD3⁺ gate was used to exclude γδ2 T cells (top panels). A second gate on CD235a⁺ RBCs and beads was used to analyze CD235a⁺CFSE⁺ RBCs (bottom panels). RBC lysis was calculated as the ratio between CFSE⁺ cells to counting beads and then normalized to the ratio in samples without γδ2 T cells.

Extended Data 3. BTN3A1 and BTN2A1 expression on iRBCs

a, Pf mixed stage culture and γδ2 T cells were stained with anti-BTN3A1, anti-BTN2A1 and Hoechst dye (DNA). Parasite stage gates were set based on RBC DNA content (rings, R; trophozoites, T; schizonts, S). b, BTN3A1 expression was plotted as median of fluorescence intensity (MFI) (n=3). c, BTN2A1 MFI in uRBCs, iRBCs, uRBCs incubated with HMBPP and γδ2 T cells (n=3). d, BTN2A1 MFI on uRBCs compared to iRBCs at different parasite stages. Isotype-stained control samples were a mixture of uRBCs, iRBCs and γδ2 T cells (n=3). In b-d, shown at left are representative histograms from 1 sample and at right are the mean ± s.e.m. of 3 independent experiments. Iso, isotype control. n, biological independent samples. Statistical analysis was by one-way ANOVA with Tukey’s multiple comparisons test. P value: **<0.01, ***<0.001, ****<0.0001. Data shown are representative of at least three independent experiments

Extended Data 4. Gating strategy to measure γδ2 T cell degranulation and parasite reinvasion

a, To measure degranulation, γδ2 T cells were co-cultured with RBCs in the presence of anti-CD107a for 4 hr. Cells were then stained with viability Live/Dead dye and anti-CD3. Single live cells were gated on SSC-A vs FSC-A, excluding dead cells. CD107a⁺ staining was analyzed on gated CD3⁺ γδ2 T cells. b, To determine the effect of γδ2 T cells on parasite reinvasion, synchronized iRBCs infected 12, 30 or 42 hr earlier were cultured for 42, 24 and 12 hr, respectively, with or without γδ2 T cells at different E:T ratios. Parasite reinvasion was measured by flow cytometry using SYBR green staining to detect parasite DNA and anti-CD235a for RBC gating and anti-CD3 to exclude γδ2 T cells. The DNA content of iRBCs at different stages enabled gating on each stage of parasite infection to quantify the proportion of iRBCs at each stage. Reinvasion of fresh RBC increased the proportion of rings. The reinvasion % was calculated as the percentage of newly invaded RBCs at ring stage in comparation with the Plasmodium culture without γδ2 T cells or any treatment (100% reinvasion).

Extended Data 5. γδ1 T cells and freshly isolated healthy donor peripheral blood γδ2 T cells do not respond to iRBCs

a, Vδ1 and Vδ2 T cells, enriched by positive selection from 3 HD and cultured for 5 days in medium containing IL-2 and IL-15, were co-cultured with uRBCs or iRBCs or no added cells in the presence of anti-CD107a. Cell degranulation was measured by CD107a staining. b-e, Highly purified freshly isolated HD γδ2 T cells from 3 donors were added to uRBCs or iRBCs to assess degranulation by CD107a staining (b), RBC lysis (c) and phagocytosis of CFSE-labeled and Pf serum-opsonized RBC (d,e). Representative images are shown in (d) and quantification of 2 independent experiments is shown in (e). Scale bar: 7 μm (d). Statistical analysis was by one-way ANOVA (a,b), two-way ANOVA with Tukey’s multiple comparisons test (c) and two-tailed nonparametric paired t-test (e). Mean ± s.e.m. is shown. P value: ***<0.001, ****<0.0001. Data shown are representative of at least three independent experiments.

Extended Data 6. iRBC lysis and parasite killing at different stages of infection by purified cytotoxic granule proteins

a, Electron microscopy of a ring stage iRBC treated or not with GzmB, PFN and GNLY showing disruption of morphology after treatment (right). In (a), parasitophorous vacuole (PV) detachment is indicated by a black arrow and increased parasite vacuolization by a red arrow. Parasite nucleus (N), hemoglobin vacuole (Hb), hemozoin (H). b, Imaging flow cytometry gating strategy for parasite developmental stages based on DNA content by Hoechst staining. M merozoites, R rings, T trophozoites, S schizonts. c, GzmB-AF488 uptake in the presence or absence of GNLY. Scale bar: 500 nm (a). n, biological independent samples. Data shown are representative of at least three independent experiments.

Figure 1.. γδ2 T cells are activated in P. falciparum infection

a, Frequency of circulating lymphocytes from 8 healthy donors (HD) and 8 P. falciparum patients (Pf). CD3⁺CD4⁺ (CD4), CD3⁺CD8⁺ (CD8), CD3⁺TCRδ2⁺ (γδ2), CD3⁻CD56⁺ (NK). b,c, Circulating γδ2 T cell activation in 8 HD and 5 Pf was evaluated by CD69 and CD16 staining. d, Intracellular granulysin (GNLY), perforin (PFN) and granzyme B (GzmB) in 8 HD and 8 Pf. e, CFSE-stained iRBC were co-cultured with fresh purified γδ2 T cells from 3 HD and 3 Pf at indicated E:T ratios for 12 hr and iRBC lysis was assessed by flow cytometry analysis of the number of viable CFSE⁺ cells relative to added counting beads. f, Plasma GNLY concentration in 16 HD and 22 Pf patients by GNLY ELISA. Statistical analysis was performed by two-tailed nonparametric unpaired t-test (a,f), two-way ANOVA with Sidak’s multiple comparisons test (d), and area under the curve, followed by two-tailed nonparametric paired t-test, followed by two-tailed nonparametric paired t-test (e). Mean ± s.e.m. is shown. P value: *<0.05, **<0.01, ***<0.001, ****<0.0001.

Figure 2.. iRBCs activate γδ2 T cells

a-c, PBMC from HD were evaluated ex vivo (day 0) and after co-culture with uRBCs or iRBCs for 7 days. Shown are representative flow cytometry histograms (left) and percent positive cells for 4 HD samples (right) after staining for cell surface CD3⁺CD4⁺ (CD4), CD3⁺CD8⁺ (CD8), CD3⁺TCRδ2⁺ (γδ2), CD3⁻CD56⁺ (NK) and intracellular GNLY (a), PFN (b) or GzmB (c). The vertical gating lines were drawn based on the unstained sample. d,e, γδ2 T cell activation before and during co-culture with uRBCs or iRBCs was evaluated by CD69 and CD16 staining, n=3 independent donor samples. Statistical analysis was performed by two-way ANOVA with Sidak’s multiple comparisons test (a-c) and two-way ANOVA with Tukey’s multiple comparisons test (d-e). Mean ± s.e.m. is shown. P value: *<0.05, **<0.01,***<0.001, ****<0.0001. Data shown are representative of three independent experiments.

Figure 3.. γδ2 T cells recognize and lyse iRBCs

a-d, Cell surface BTN3A1 on RBCs (a, top row) and γδ2 T cells (a, bottom) imaged and quantified for 3 HD samples (b) by confocal microscopy and by flow cytometry (c,d). DAPI stains parasite DNA in iRBCs and T cell nuclei; CD235A stains glycophorin A on RBCs. An isotype control antibody (Iso) was added to a mixture of uRBCs, iRBCs, γδ2 T cells (c,d). e-i, Highly purified HD γδ2 T cells activated by culture in IL-2 and IL-15, were added to uRBCs or iRBCs to assess immunological synapse formation (e) and conjugation with uRBCs and iRBCs by imaging flow cytometry (n=6) (f), degranulation by externalized CD107a (n=5 and 8) (g), RBC lysis by flow cytometry (n=5) (h) and effect on parasite reinvasion (n=5) (i). (e) shows representative images of conjugated cells by bright field (BF) and staining for LFA-1, BTN3A1 (BTN3), CD3 and Hoechst DNA dye. In (i), parasite reinvasion was measured by flow cytometry after iRBCs synchronized at ring, trophozoite and schizont stages were cultured with or without γδ2 T cells at indicated E:T ratios. j,k, γδ2 T cells and RBCs, cultured together or separated by a Transwell membrane (n=4), were assayed for γδ2 T cell degranulation by CD107a staining (j) and RBC lysis (k). l, γδ2 T cells, pre-incubated or not with EGTA, were added to CFSE-labeled RBCs and RBC lysis was measured by flow cytometry (n=4). Scale bars: 10 μm (a), 7 μm (e). n, biological independent samples. Statistical analysis was by one-way ANOVA with Tukey’s multiple comparisons test (b,d,g), two-tailed nonparametric unpaired t-test (f), and area under the curve, followed by two-tailed nonparametric paired t-test (h), area under the curve, followed by one-way ANOVA (k) or two-way ANOVA with Sidak’s multiple comparisons test (i,j,l). Mean ± s.e.m. is shown. P value: *<0.05, **<0.01, ***<0.001, ****<0.0001. Data shown are representative of three independent experiments.

Figure 4.. GNLY delivers GzmB into iRBCs to cause iRBC lysis and parasite killing

a-c, RBCs, incubated with indicated combinations of cytotoxic effector proteins (n=3), were assayed for RBC lysis by LDH release (a), parasite reinvasion into fresh RBCs by flow cytometry (b), and parasite morphology by electron microscopy (c,d). (c) quantifies prominent morphological alterations in EM images - parasitophorous vacuole (PV) detachment (black arrows) and increased parasite vacuolization (red arrows) in 15 images from 3 sections. Parasite nucleus (N), food vacuole (FV), hemozoin (Hz) are labeled in untreated iRBCs. e, uRBCs or unsynchronized iRBCs, stained with 25-NBD or filipin as cholesterol probes and Hoechst for parasite DNA, were analyzed by imaging flow cytometry using DNA staining to gate for different parasite stages (R ring, T trophozoite, S schizont). BF, bright field, H, Hoechst dye. f, Representative histograms (top) and quantification of multiple samples (bottom) of AF647-GNLY binding to uRBCs and iRBCs (trophozoite stage) by flow cytometry (n=5). g,h, GzmB-AF488 internalization in the absence or presence of GNLY or PFN assessed by imaging flow cytometry (n=3). (g) shows GzmB-AF488 (Gzm) internalization in representative images and (h) shows the proportion of RBCs at different parasite stages that stained for GzmB in multiple experiments, n=3. i, Representative images (left) and quantification (right) of imaging flow cytometry showing RBC, uninfected or infected at indicated parasite stage, incubated with GzmB-AF488, GNLY-AF647 and unlabeled GNLY, n=3. M, merozoite; R, ring; T, trophozoite; S, schizont; H, Hoechst dye; BF, bright field; n, biological independent samples. Scale bars: 500 nm (d), 7 μm (e,g,i). Statistical analysis was by one-way ANOVA with Tukey’s multiple comparisons test (a,b,c,h,i) or two-tailed nonparametric unpaired t-test (f). Mean ± s.e.m. is shown. P value: *<0.05, **<0.01, ***<0.001, ****<0.0001. Data shown are representative of three independent experiments.

Figure 5.. P. falciparum HMBPP activates γδ2 T cells via the γδTCR and BTN3A1

a,b, Highly purified HD γδ2 T cells, pre-activated with IL-2 and IL-15, were co-cultured with iRBCs in the presence of indicated blocking or isotype control (Iso) antibodies (n=4) and assayed for γδ2 T cell degranulation by cell surface CD107a (a) and RBC lysis (b) by flow cytometry. c,d, HD γδ2 T cells were co-cultured with iRBCs pre-treated with indicated combinations of fosmidomycin and IPP, uRBCs or HMBPP to assess degranulation (n=3) (c) and RBC lysis (n=5) (d). e,f, γδ2 T cell or iRBCs were pre-incubated with blocking antibodies to γδTCR, BTN3A1 or isotype control (Iso) before co-culture (n=3) to assess degranulation (e) and RBC lysis (f). n, biological independent samples. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test. Mean ± s.e.m. is shown; P value: *<0.05, **<0.01, ***<0.001, ****<0.0001. Data shown are representative of three independent experiments.

Figure 6.. γδ2 T cells phagocytose opsonized P. falciparum-infected RBCs

a,b, CFSE-stained iRBCs and uRBCs, pre-incubated with HD human AB serum, Pf-infected patient serum or anti-CD235a, were co-cultured with HD γδ2 T cells and assessed for phagocytosis by costaining of gated singlet cells for CFSE and CD3 by imaging flow cytometry. Representative images of three experiments are shown in (a) and quantification of the proportion of γδ2 T cells with internalized RBCs in 3 samples is in (b). c,d, CFSE-labeled iRBC were opsonized with Pf serum and co-cultured with 3 HD γδ2 T cells. Samples were fixed and stained with PerCP-Cy5.5-anti-CD235a and PB-anti-CD3. (c) shows representative images of engulfed iRBC (CFSE⁺CD235a⁻) by γδ2 T cells (CD3⁺) (top 2 panels) and free iRBCs (CFSE⁺CD235a⁺) (bottom 2 panels), quantified in (d) for the proportion of cells that stained for accessible RBC marker CD235a. e, Confocal time-lapse images (63x) over 1 hr of co-culture of CellTracker-labeled γδ2 T cells (red) and CSFE-labeled iRBCs (green) opsonized with Pf serum (left) and quantification of proportion of γδ2 T cells that internalized iRBCs (upper right). Magnified 3D reconstructions of individual cells in two orthogonal rotations at 30’ and 60’ are shown at right. f,g, Effect of indicated blocking antibodies on HD γδ2 T cell phagocytosis (f) and inhibition of parasite reinvasion (g). γδ2 T cells were pre-treated with blocking or isotype (Iso) control antibodies before co-culture with CFSE-stained, Pf serum-opsonized iRBCs. Phagocytosis was measured by imaging flow cytometry and parasite reinvasion was measured by flow cytometry. h, Effect of an actin inhibitor and Ca⁺⁺ chelator on γδ2 T cell lysis (left) and phagocytosis (right) of iRBCs opsonized with Pf serum (n=4). HD γδ2 T cells were pre-treated with EGTA or cytochalasin D (CytD) prior to co-culture with iRBCs. i,j, Comparison of the effect of HD and Pf sera on HD γδ2 T cell iRBC lysis (i, left), phagocytosis (i, right) (n=4) and inhibition of parasite reinvasion (j). n, biological independent samples. Scale bars: 7 μm (a,c), 10 μm (e). Statistical analysis was by two-way ANOVA (b,j), one-way ANOVA with Tukey’s multiple comparisons test (f-h), two-tailed Chi-square test (e), and two-tailed nonparametric unpaired t-test (d,i). Mean ± s.e.m. is shown. P value: *<0.05, **<0.01, ***<0.001, ****<0.0001. Data shown are representative of at least three independent experiments.