Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: γδ2 T cells are activated in P. falciparum infection.
Fig. 2: iRBCs activate γδ2 T cells.
Fig. 3: γδ2 T cells recognize and lyse iRBCs.
Fig. 4: GNLY delivers GzmB into iRBCs to cause iRBC lysis and parasite killing.
Fig. 5: P. falciparum HMBPP activates γδ2 T cells via the γδTCR and BTN3A1.
Fig. 6: γδ2 T cells phagocytose opsonized P. falciparum–infected RBCs.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request.

References

  1. 1.

    Junqueira, C. et al. Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax-infected reticulocytes. Nat. Med 24, 1330–1336 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Silvestre, D., Kourilsky, F. M., Nicolai, M. G. & Levy, J. P. Presence of HLA antigens on human reticulocytes as demonstrated by electron microscopy. Nature 228, 67–68 (1970).

    CAS  PubMed  Google Scholar 

  3. 3.

    Worku, S. et al. Lymphocyte activation and subset redistribution in the peripheral blood in acute malaria illness: distinct γδ+ T cell patterns in Plasmodium falciparum and P. vivax infections. Clin. Exp. Immunol. 108, 34–41 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Troye-Blomberg, M. et al. Human γδ T cells that inhibit the in vitro growth of the asexual blood stages of the Plasmodium falciparum parasite express cytolytic and proinflammatory molecules. Scand. J. Immunol. 50, 642–650 (1999).

    CAS  PubMed  Google Scholar 

  5. 5.

    Ramsey, J. M. et al. Plasmodium falciparum and P. vivax gametocyte-specific exoantigens stimulate proliferation of TCR γδ+ lymphocytes. J. Parasitol. 88, 59–68 (2002).

    CAS  PubMed  Google Scholar 

  6. 6.

    Pichyangkul, S., Saengkrai, P., Yongvanitchit, K., Stewart, A. & Heppner, D. G. Activation of γδ T cells in malaria: interaction of cytokines and a schizont-associated Plasmodium falciparum antigen. J. Infect. Dis. 176, 233–241 (1997).

    CAS  PubMed  Google Scholar 

  7. 7.

    Jagannathan, P. et al. Vδ2+ T cell response to malaria correlates with protection from infection but is attenuated with repeated exposure. Sci. Rep. 7, 11487 (2017).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Farrington, L. A. et al. Frequent malaria drives progressive Vδ2 T-cell loss, dysfunction, and CD16 up-regulation during early childhood. J. Infect. Dis. 213, 1483–1490 (2016).

    CAS  PubMed  Google Scholar 

  9. 9.

    Jagannathan, P. et al. Loss and dysfunction of Vδ2+ γδ T cells are associated with clinical tolerance to malaria. Sci. Transl. Med. 6, 251ra117 (2014).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Farouk, S. E., Mincheva-Nilsson, L., Krensky, A. M., Dieli, F. & Troye-Blomberg, M. γδ T cells inhibit in vitro growth of the asexual blood stages of Plasmodium falciparum by a granule exocytosis-dependent cytotoxic pathway that requires granulysin. Eur. J. Immunol. 34, 2248–2256 (2004).

    CAS  PubMed  Google Scholar 

  11. 11.

    Liu, C. et al. Vγ9Vδ2 T cells proliferate in response to phosphoantigens released from erythrocytes infected with asexual and gametocyte stage Plasmodium falciparum. Cell. Immunol. 334, 11–19 (2018).

    CAS  PubMed  Google Scholar 

  12. 12.

    Guenot, M. et al. Phosphoantigen burst upon Plasmodium falciparum schizont rupture can distantly activate Vγ9Vδ2 T cells. Infect. Immun. 83, 3816–3824 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Vavassori, S. et al. Butyrophilin 3A1 binds phosphorylated antigens and stimulates human γδ T cells. Nat. Immunol. 14, 908–916 (2013).

    CAS  PubMed  Google Scholar 

  14. 14.

    Wang, H. et al. Butyrophilin 3A1 plays an essential role in prenyl pyrophosphate stimulation of human Vγ2Vδ2 T cells. J. Immunol. 191, 1029–1042 (2013).

    CAS  PubMed  Google Scholar 

  15. 15.

    Seder, R. A. et al. Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science 341, 1359–1365 (2013).

    CAS  PubMed  Google Scholar 

  16. 16.

    Mordmuller, B. et al. Sterile protection against human malaria by chemoattenuated PfSPZ vaccine. Nature 542, 445–449 (2017).

    PubMed  Google Scholar 

  17. 17.

    Ishizuka, A. S. et al. Protection against malaria at 1 year and immune correlates following PfSPZ vaccination. Nat. Med. 22, 614–623 (2016).

    CAS  PubMed  Google Scholar 

  18. 18.

    Zaidi, I. et al. γδ T cells are required for the induction of sterile immunity during irradiated sporozoite vaccinations. J. Immunol. 199, 3781–3788 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Costa, G. et al. Control of Plasmodium falciparum erythrocytic cycle: γδ T cells target the red blood cell-invasive merozoites. Blood 118, 6952–6962 (2011).

    CAS  PubMed  Google Scholar 

  20. 20.

    Dotiwala, F. & Lieberman, J. Granulysin: killer lymphocyte safeguard against microbes. Curr. Opin. Immunol. 60, 19–29 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Wu, Y. et al. Human γδ T cells: a lymphoid lineage cell capable of professional phagocytosis. J. Immunol. 183, 5622–5629 (2009).

    CAS  PubMed  Google Scholar 

  22. 22.

    Himoudi, N. et al. Human γδ T lymphocytes are licensed for professional antigen presentation by interaction with opsonized target cells. J. Immunol. 188, 1708–1716 (2012).

    CAS  PubMed  Google Scholar 

  23. 23.

    Brandes, M., Willimann, K. & Moser, B. Professional antigen-presentation function by human γδ T cells. Science 309, 264–268 (2005).

    CAS  PubMed  Google Scholar 

  24. 24.

    Meuter, S., Eberl, M. & Moser, B. Prolonged antigen survival and cytosolic export in cross-presenting human γδ T cells. Proc. Natl Acad. Sci. USA 107, 8730–8735 (2010).

    CAS  PubMed  Google Scholar 

  25. 25.

    Howard, J. et al. The antigen-presenting potential of Vγ9Vδ2 T cells during Plasmodium falciparum blood-stage infection. J. Infect. Dis 215, 1569–1579 (2017).

    CAS  PubMed  Google Scholar 

  26. 26.

    Barisa, M. et al. E. coli promotes human Vγ9Vδ2 T cell transition from cytokine-producing bactericidal effectors to professional phagocytic killers in a TCR-dependent manner. Sci. Rep. 7, 2805 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Hsiao, C. H. et al. Synthesis of a phosphoantigen prodrug that potently activates Vγ9Vδa2 T-lymphocytes. Chem. Biol. 21, 945–954 (2014).

    CAS  PubMed  Google Scholar 

  28. 28.

    Yang, Y. et al. A structural change in butyrophilin upon phosphoantigen binding underlies phosphoantigen-mediated Vγ9Vδ2 T cell activation. Immunity 50, 1043–1053.e5 (2019).

    CAS  PubMed  Google Scholar 

  29. 29.

    Gu, S. et al. Phosphoantigen-induced conformational change of butyrophilin 3A1 (BTN3A1) and its implication on Vγ9Vδ2 T cell activation. Proc. Natl Acad. Sci. USA 114, E7311–E7320 (2017).

    CAS  PubMed  Google Scholar 

  30. 30.

    Karunakaran, M. M. et al. Butyrophilin-2A1 directly binds germline-encoded regions of the Vγ9Vδ2 TCR and is essential for phosphoantigen sensing. Immunity 52, 487–498.e6 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Rigau, M. et al. Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδ T cells. Science https://doi.org/10.1126/science.aay5516 (2020).

  32. 32.

    Dotiwala, F. et al. Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites. Nat. Med. 22, 210–216 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Muto, M., Baghdadi, M., Maekawa, R., Wada, H. & Seino, K. Myeloid molecular characteristics of human γδ T cells support their acquisition of tumor antigen-presenting capacity. Cancer Immunol. Immunother. 64, 941–949 (2015).

    CAS  PubMed  Google Scholar 

  34. 34.

    Yeh, E. & DeRisi, J. L. Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum. PLoS Biol. 9, e1001138 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Kurup, S. P., Butler, N. S. & Harty, J. T. T cell-mediated immunity to malaria. Nat. Rev. Immunol. 19, 457–471 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Arora, G. et al. NK cells inhibit Plasmodium falciparum growth in red blood cells via antibody-dependent cellular cytotoxicity. Elife https://doi.org/10.7554/eLife.36806 (2018).

  37. 37.

    Goodier, M. R., Wolf, A. S. & Riley, E. M. Differentiation and adaptation of natural killer cells for anti-malarial immunity. Immunol. Rev. 293, 25–37 (2020).

    CAS  PubMed  Google Scholar 

  38. 38.

    Ravenhill, B. J. et al. Quantitative comparative analysis of human erythrocyte surface proteins between individuals from two genetically distinct populations. Commun. Biol. 2, 350 (2019).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Bryk, A. H. & Wisniewski, J. R. Quantitative analysis of human red blood cell proteome. J. Proteome Res. 16, 2752–2761 (2017).

    CAS  PubMed  Google Scholar 

  40. 40.

    Barbee, S. D. et al. Skint-1 is a highly specific, unique selecting component for epidermal T cells. Proc. Natl Acad. Sci. USA 108, 3330–3335 (2011).

    CAS  PubMed  Google Scholar 

  41. 41.

    Turchinovich, G. & Hayday, A. C. Skint-1 identifies a common molecular mechanism for the development of interferon-γ-secreting versus interleukin-17-secreting γδ T cells. Immunity 35, 59–68 (2011).

    CAS  PubMed  Google Scholar 

  42. 42.

    Gordon, S. Phagocytosis: an immunobiologic process. Immunity 44, 463–475 (2016).

    CAS  PubMed  Google Scholar 

  43. 43.

    Chien, Y. H., Meyer, C. & Bonneville, M. γδ T cells: first line of defense and beyond. Annu. Rev. Immunol. 32, 121–155 (2014).

    CAS  PubMed  Google Scholar 

  44. 44.

    Das, S. et al. Evolution of two prototypic T cell lineages. Cell Immunol. 296, 87–94 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Barry, A. & Hansen, D. Naturally acquired immunity to malaria. Parasitology 143, 125–128 (2016).

    PubMed  Google Scholar 

  46. 46.

    Crompton, P. D. et al. Malaria immunity in man and mosquito: insights into unsolved mysteries of a deadly infectious disease. Annu. Rev. Immunol. 32, 157–187 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Moebius, J. et al. PD-1 expression on NK cells in malaria-exposed individuals is associated with diminished natural cytotoxicity and enhanced antibody dependent cellular cytotoxicity. Infect. Immun. 88, e00711-19 (2020).

    PubMed  PubMed Central  Google Scholar 

  48. 48.

    Hart, G. T. et al. Adaptive NK cells in people exposed to Plasmodium falciparum correlate with protection from malaria. J. Exp. Med. 216, 1280–1290 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Hernandez-Castaneda, M. A. et al. γδ T cells kill Plasmodium falciparum in a granzyme- and granulysin-dependent mechanism during the late blood stage. J. Immunol. 204, 1798–1809 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Stenger, S. et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 282, 121–125 (1998).

    CAS  PubMed  Google Scholar 

  51. 51.

    Ribaut, C. et al. Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species. Malar. J. 7, 45 (2008).

    PubMed  PubMed Central  Google Scholar 

  52. 52.

    Betts, M. R. et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J. Immunol. Methods 281, 65–78 (2003).

    CAS  PubMed  Google Scholar 

  53. 53.

    Thiery, J., Walch, M., Jensen, D. K., Martinvalet, D. & Lieberman, J. Isolation of cytotoxic T cell and NK granules and purification of their effector proteins. Curr. Protoc. Cell Biol. 47, 3.37.1–3.37.29 (2010).

    Google Scholar 

Download references

Acknowledgements

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).

Author information

Affiliations

Authors

Contributions

This study was conceived by J.L., C.J., R.B.P., S.A. and J.D.D., and experiments were performed by C.J. and R.B.P. with help from G.C., S.A., Z.L., S.S.S. and A.C. D.B.P. and R.T.G. recruited patients infected with Pf and healthy donors. C.J. and R.T.G. supervised endemic area field study. All authors contributed to data analysis. J.L. and C.J. wrote the manuscript.

Corresponding authors

Correspondence to Caroline Junqueira or Judy Lieberman.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

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 CD3CD56+ (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 Fig. 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 Fig. 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 Fig. 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 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.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing