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.
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This research was supported by NIH AI116577 and AI131632 (J.L. and R.T.G.), NIH U19 AI089681 Amazonian Center of Excellence in Malaria Research (C.J. and R.T.G.), NIH AI145941 (J.D.D.), Harvard University Lemann Brazil Fund (J.L. and C.J.) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais-FAPEMIG APQ-00653-16 (C.J.), Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP, 2016/23618-8 (C.J. and R.T.G.), Brazilian National Institute of Science and Technology for Vaccines (CNPq/FAPMIG) (C.J. and R.T.G.). C.J., R.B.P., G.C. and R.T.G. are recipients of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) fellowships. C.J. and G.C. are fellows of Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES).
The authors declare no competing interests.
Peer review information Nature Immunology thanks Robert Sauerwein and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 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.
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.
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.
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 Fig. 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 Fig. 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.
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Junqueira, C., Polidoro, R.B., Castro, G. et al. γδ T cells suppress Plasmodium falciparum blood-stage infection by direct killing and phagocytosis. Nat Immunol 22, 347–357 (2021). https://doi.org/10.1038/s41590-020-00847-4
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