PMC

LFA-1 is involved in NK cell interaction with and killing of iRBCs. (A) NK cells were cultured with RBCs and 3D7 schizonts in the presence or absence of the indicated blocking antibodies, and parasitemia was quantified by flow cytometry 96 h later. Shown are representative CD56- vs. Hoechst-staining profiles of cells. The numbers indicate the percentage of iRBCs among total RBCs. (B and C) NK cells were cultured with RBCs and 3D7 schizonts in the presence or absence of the LFA-1–blocking antibody. (B) The culture was visualized by video microscopy. Shown are the lengths of time NK cells interact with iRBCs with (αLFA-1) and without (NK+iRBCs) the blocking antibody. The data shown are the average of 20 NK cells that interacted with iRBCs. (C) NK cells were assayed for CD69 expression at 48 h. Shown is CD69 expression by CD56⁺ NK cells in the presence or absence of the LFA-1–blocking antibody. The numbers indicate percentages of CD69⁺ cells.

Cell–cell contact is required for NK cell activation and killing of iRBCs. (A and B) Human RBCs were infected with purified 3D7 schizonts in the absence or presence of human NK cells in the same well or in a separate compartment in the transwell. NK cells were assayed for CD69 expression at 4, 12, and 24 h, and parasitemia was measured at 96 h by flow cytometry. (A) Comparison of CD69 expression by CD56⁺ NK cells in the same well or in a separate compartment in the transwell at the indicated time points of culture. The numbers indicate percentages of CD69⁺ cells. (B) Representative CD56- vs. Hoechs- staining profiles of cultured cells at 96 h. (C) Transwell assay of the killing of iRBCs by NK cells. NK cells were cultured with RBCs and 3D7 schizonts in one chamber (w/ NK). The other chamber contained only RBCs and parasites (w/o NK). Parasitemia in both chambers was quantified by flow cytometry at 48 and 96 h. Shown are representative CD56- vs. Hoechst-staining profiles of cells from the two chambers. The numbers in B and C indicate the percentage of iRBCs among total RBCs.

NK cells play a critical role in the control of parasite infection. Seven days after cytokine treatment and 6 d after RBC supplementation, RICH mice were injected with PBS, anti-human CD56 antibody (αCD56), or anti-human CD14 antibody (αCD14). One day later, mice were bled, and the levels of human NK cells and monocytes/macrophages in PBMCs were analyzed by flow cytometry. Then mice were infected immediately with 5 × 10⁶ 3D7 ring-stage parasites. Two days later, parasitemia was analyzed in the whole blood by microscopy, and human IFN-γ and IL-6 in the serum were measured by ELISA. (A) Depletion of human CD56⁺ NK cells and CD14⁺ monocytes/macrophages in treated mice. Shown are representative CD14 vs. CD56 staining profiles of hCD45⁺ PBMCs of control, αCD56-, and αCD14-treated mice. The numbers indicate the percentage of cells in the gated regions. (B–D) Comparison of parasitemia (B) and serum levels of human IFN-γ (C) and IL-6 (D) in the three groups of mice. Each symbol represents one mouse. Data represent mean ± SEM. *P < 0.01. n = 6 for each group.

RICH mice support a robust P. falciparum infection. Humanized mice were injected hydrodynamically with plasmids encoding human IL-15 (50 µg) and Flt3L (10 µg) on day zero. Beginning 1 d after plasmid injection, mice were injected i.p. daily with 1 mL human RBCs. After 7 d of RBC supplementation, mice were infected i.v. with 5 × 10⁶ ring-stage 3D7 parasites and given daily RBC supplementation throughout the experiment (n = 10). (A–C) Human RBC and immune cell reconstitution before parasite infection (7 d after plasmid injection). Shown are representative CD235ab vs. forward scatter (FSC) profiles of whole blood (A), human CD45 (hCD45) vs. mouse CD45.1 (mCD45) staining profiles of PBMCs (B), and human CD56 vs. CD14 and CD3 vs. CD19 staining profiles gating on human CD45⁺ cells (C). The numbers indicate the percentage of cells in the gated regions. The mean ± SEM (n = 10) are shown in Results. (D) Quantification of parasitemia in RICH mice at different time points. Data are shown as percentage (mean ± SEM) of iRBCs among total RBCs and are a compilation from two independent experiments with five mice in each experiment.

Human NK cells respond to and eliminate iRBCs in vitro. Human RBCs were incubated with purified 3D7 schizonts in the presence or absence of purified human NK cells. Some cultures were visualized by video microscopy to follow NK cell migration and interaction with RBCs. Some cultures were stained for CD69 to monitor NK cell activation or were stained by Hoechst plus anti-CD56 to quantify parasitemia. (A) Comparison of the lengths of time during which NK cells interact with infected vs. uninfected RBCs. The data are averages of 100 NK cells interacting with infected or uninfected RBCs. (B) Comparison of CD69 expression by CD56⁺ NK cells at the indicated time points of culture. The numbers indicate the percentages of CD69⁺ cells. (C) Representative CD56- vs. Hoechst-staining profiles of cultured cells at 48 and 96 h. (D) Humanized mice were hydrodynamically injected with plasmids encoding human IL-15 and Flt3L to enhance reconstitution of human NK cells. Seven days later, NK cells were purified from blood, spleen, lung, and liver and were used in the culture as above. Shown are CD56- vs. Hoechst-staining profiles of cultures with or without NK cells at 48 and 96 h. (E) NK cells were cultured with RBCs and 3D7 schizonts in the presence or absence of CMA. Parasitemia was measured by flow cytometry 96 h after coculture. Representative CD56- vs. Hoechst-staining profiles of cultured cells are shown. The numbers in B– E indicate the percentages of iRBCs among total RBCs.