Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax-infected reticulocytes

Abstract

Plasmodium vivax causes approximately 100 million clinical malaria cases yearly^1,2. The basis of protective immunity is poorly understood and thought to be mediated by antibodies^3,4. Cytotoxic CD8⁺ T cells protect against other intracellular parasites by detecting parasite peptides presented by human leukocyte antigen class I on host cells. Cytotoxic CD8⁺ T cells kill parasite-infected mammalian cells and intracellular parasites by releasing their cytotoxic granules^5,6. Perforin delivers the antimicrobial peptide granulysin and death-inducing granzymes into the host cell, and granulysin then delivers granzymes into the parasite. Cytotoxic CD8⁺ T cells were thought to have no role against Plasmodium spp. blood stages because red blood cells generally do not express human leukocyte antigen class I⁷. However, P. vivax infects reticulocytes that retain the protein translation machinery. Here we show that P. vivax-infected reticulocytes express human leukocyte antigen class I. Infected patient circulating CD8⁺ T cells highly express cytotoxic proteins and recognize and form immunological synapses with P. vivax-infected reticulocytes in a human leukocyte antigen-dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. P. vivax-infected reticulocytes and parasite killing is perforin independent, but depends on granulysin, which generally efficiently forms pores only in microbial membranes⁸. We find that P. vivax depletes cholesterol from the P. vivax-infected reticulocyte cell membrane, rendering it granulysin-susceptible. This unexpected T cell defense might be mobilized to improve P. vivax vaccine efficacy.

Figure 1. Increased frequency of activated CD8⁺ T cells in the peripheral blood of P. vivax patients

Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD) and P. vivax malaria patients (Pv) were gated on CD8⁺CD3⁺ T cells (gating strategy described in Supplementary Fig. 1) and analyzed for expression of activation markers and cytotoxic granule proteins by flow cytometry. a,b, Shown are representative flow plots (a) and the proportion of CD8⁺ T cells expressing CD69, HLA-DR and Ki67 (b) malaria patients, before treatment (BT) and 30–40 days after treatment and parasitological cure (AT). n=8 biologically independent samples/independent experiments. Statistical analysis was performed by two-tailed parametric paired t-test at 95% confidence interval (CI). c,d, Shown are representative flow plots (c) and the proportion of peripheral blood CD8⁺ T cells expressing GzmB, PFN, and GNLY (d) HD and P. vivax (Pv) malaria patients BT. n=5 biologically independent samples/independent experiments. e, The levels of soluble GNLY in plasma of n=7 HD and n=10 Pv patients BT were measured by ELISA as biologically independent samples/independent experiments. (d,e) show mean ± SEM; statistical analysis was performed by two-tailed non-parametric unpaired t-test at 95% CI.

Figure 2. Increased HLA-ABC in P. vivax infected reticulocytes

a, Gating strategy to evaluate P. vivax infection and HLA-expression in reticulocytes. Top and bottom panels are representative results from a healthy donor (HD) and P. vivax acute malaria patient (Pv) before treatment (BT), respectively. Retics are CD71⁺CD235a⁺ and SYBR Green detects parasite DNA in iRetics. A pan-HLA class I antibody was used to analyze HLA expression. This experiment was repeated four times with similar results. b–d, Comparison of percent of retics in RBC gate (b), percent of Retics that express HLA-I, (c) percent of SYBR Green⁺ iRetics (d) in blood from HD (n=8) and Pv BT patients (n=8). Shown are mean ± SEM; statistical analysis by two-tailed non-parametric unpaired t-test at 95% CI. e, Comparison of HLA-ABC expression in circulating uRetics and iRetics in n=8 Pv BT samples, based on SYBR Green I and HLA staining of CD235a⁺CD71⁺ Retics, representative dot plot in (a). Shown are mean ± SEM; statistical analysis used a two-tailed parametric paired t-test at 95% CI. f,g, Comparison of HLA-ABC (f) and HLA-DR (g) expression by HD uRetics and Pv iRetics and CD19⁺ B cells. Light gray histograms are unstained and darker gray histograms are stained cells. Shown are representative samples of 5 analyzed. h, Imaging flow cytometry of representative Pv uRetics (top) and iRetics (bottom) stained for CD235a, SYBR Green, CD71, HLA-ABC and HLA-DR. This experiment was repeated three times with similar results. i, Immunoblot of cell lysates from 3 HD uRetics and 3 Pv iRetics, loaded with 50 μg of protein per well and probed for the antigen processing protein, TAP1 as well as β-actin and F-actin as loading controls for HD uRetics and Pv BT iRetics, respectively. HD PBMCs were used as a positive control (20 μg). This experiment was repeated three times with similar results.

Figure 3. CD8⁺ T cells are activated by and lyse autologous P. vivax-infected reticulocytes

a–d, Purified CD8⁺ or CD4⁺ T lymphocytes from healthy donors (HD) or P. vivax malaria patients (Pv) before treatment (BT) were cultured in medium alone, with autologous uninfected Retics (uRetics), with purified infected Retics (iRetics), or in the presence anti-CD3 and anti-CD28 and analyzed by flow cytometry for the proportion of CD8⁺ T cells staining for CD69 (a) and Ki67 (b) (n=5) or intracellular IFNγ (c) (n=8); or CD4⁺ T cells staining for intracellular IFNγ (d) (n=6). e, IFNγ expression by CD8⁺ T cells after stimulation with autologous uninfected RBC (uRBC), purified iRetic or anti-CD3 and anti-CD28 in the presence of anti-HLA-ABC or isotype control antibody (n=6). f,g, Imaging flow cytometry analysis of immune synapse formation between CD8⁺ T cells and autologous purified iRetics from Pv patients or uRetics from HD (n=5). Shown are representative images of immunological synapses between CD8⁺ T cells and purified iRetics from a Pv sample (f) and mean ± SEM of the proportion of CD8⁺ T cells forming synapses in 5 HD samples with autologous uRetics and in 5 Pv patient BT samples with purified iRetics (g). n=5 biologically independent samples/independent experiments. Cells were stained for TCR and CD235a, CD3, HLA-ABC, and CD8 and synapses were identified by the capping and co-localization of TCR, CD3, CD8 and HLA-ABC where the T cell and RBC are juxtaposed. The enlarged overlay image on the right corresponds to the bottom image. h,i, Survival of iRetics after 12 hr incubation with medium or autologous CD8⁺ T cells from Pv samples (n=5) (h) or of uRetics incubated with medium or autologous HD CD8⁺ T cells (n=4) (i), added at indicated E:T ratios, as assayed using CFSE-labeled Retics. j, iRetic lysis after 12 hr incubation with medium or autologous CD8⁺ T cells from Pv samples (n=8), measured by LDH release. k, Frequency of CD8⁺ T cells in the blood of 5 untreated Pv patients that degranulate, assessed by CD107a staining, in response to indicated stimuli. Shown are mean ± SEM; statistical analysis by non-parametric two-way ANOVA (a–e), two-tailed non-parametric unpaired t-test at 95% CI (g), and non-parametric one-way ANOVA (h–k).

Figure 4. Granulysin binds to infected reticulocytes and mediates host cell lysis and parasite killing

a–f, Imaging flow cytometry analysis of uptake of GzmB-AF488 (a,b) or GNLY-AF488 (c,d) on their own, or of GzmB-AF488 in the presence of unlabeled GNLY (e,f) by healthy donor (HD) unifected Retics (uRetics) and by uRetics and infected retics (iRetics) from acute untreated P. vivax patients (Pv). (a,c,e) show representative images, while (b,d,f) show mean ± SEM of 3 HD and Pv samples. g, Imaging flow cytometry images of Pv uRetic and iRetics incubated with GzmB-AF488 and GNLY-AF750, and stained for CD235a and with a Hoechst dye to stain parasite DNA. BF, bright field. This experiment was repeated three times with similar results. h–j, Effect of mβCD depletion of cholesterol in HD RBCs on binding of GNLY-AF488 on its own (n=4). Shown p values are in comparison to intact RBC. (h) and of GzmB-AF488 in the presence of unlabeled GNLY (n=3) (i); and on RBC lysis by increasing amounts of GNLY, assessed by LHD release (n=4). k,l, Staining of HD uRetics and of untreated Pv patient uRetics and iRetics with the cholesterol analog 25-NBD (n=3). Representative images are shown in (k) (n=5) and the proportion of cells with detectable 25-NBD fluorescence is shown in (l) (n=4). m,n, Cytolysis of Pv iRetics (m, n=5) or HD uRetics (n, n=4) after 1 hr incubation with GzmB ± GNLY ± PFN, assessed by LDH release. o, Effect of incubation of iRetics from 4 Pv patients for 1 hr with indicated cytotoxic granule proteins on parasite invasion of fresh Retics, assessed by Giemsa staining. p–r, Electron micrographs of iRetics that were untreated or incubated with GNLY ± GzmB. Higher magnification images after treatment with GNLY plus GzmB in (q) show parasitophorous vacuole membrane disruption ( ) and chromatin condensation ( ) (left); cytoplasmic vacuolization ( ) and dense granules ( ) (middle); and mitochondrial swelling ( ) (right). Higher magnification images of untreated cells (r) show intact digestive vacuole ( ), parasitophorous vacuole membrane ( ) and mitochondria ( ). These experiments were repeated three times with similar results (p,r). Graphs show mean ± SEM; statistics in (b,d,f,j,l–o) were analyzed by one-way ANOVA and in (h,i) by two-tailed non-parametric paired t-test at 95% CI.