Antibody mediated activation of natural killer cells in malaria exposed pregnant women

Abstract

Immune effector responses against Plasmodium falciparum include antibody-mediated activation of innate immune cells, which can induce Fc effector functions, including antibody-dependent cellular cytotoxicity, and the secretion of cytokines and chemokines. These effector functions are regulated by the composition of immunoglobulin G (IgG) Fc N-linked glycans. However, a role for antibody-mediated natural killer (NK) cells activation or Fc N-linked glycans in pregnant women with malaria has not yet been established. Herein, we studied the capacity of IgG antibodies from pregnant women, with placental malaria or non-placental malaria, to induce NK cell activation in response to placental malaria-associated antigens DBL2 and DBL3. Antibody-mediated NK cell activation was observed in pregnant women with malaria, but no differences were associated with susceptibility to placental malaria. Elevated anti-inflammatory glycosylation patterns of IgG antibodies were observed in pregnant women with or without malaria infection, which were not seen in healthy non-pregnant controls. This suggests that pregnancy-associated anti-inflammatory Fc N-linked glycans may dampen the antibody-mediated activation of NK cells in pregnant women with malaria infection. Overall, although anti-inflammatory glycans and antibody-dependent NK cell activation were detected in pregnant women with malaria, a definitive role for these antibody features in protecting against placental malaria remains to be proven.

Figure 1

Schematic representation of VAR2CSA and overview of cohort groups. (a) The extracellular region of VAR2CSA contains a N-terminal sequence (NTS) followed by Duffy binding-like (DBL) domains and interdomain (ID) regions. It is anchored in the membrane by a transmembrane (TM) domain connected to an acidic terminal segment (ATS). (b) Plasma samples were obtained from pregnant women in Papua New Guinea between November 2009 and August 2012 upon enrolment into an Intermittent Preventive Treatment in Pregnancy (IPTp) randomized controlled trial at 14–26 gestation weeks. Samples were grouped based on infection status at enrolment for Fc N-linked glycan profiling and grouped based on infection status at delivery for functional NK cell activation assays.

Figure 2

Gating strategy to identify NK cell activation markers. Purified NK cells were incubated with IgG test samples in presence of DBL2 or DBL3 for 5 h and then analyzed via flow cytometry. Representative flow cytometry plots of one sample to visualize gating strategy. (a) NK cells were identified by sequentially gating on lymphocytes, single cells, CD3^- cells, and NK cell subsets. NK cells subsets were gated as one and assessed for surface CD107a expression and intracellular IFNγ and TNFα production in presence of DBL2 (b) and DBL3 (c) (High response = blue; malaria-naïve response = green).

Figure 3

Human NK cells lack activation in presence of DBL2- or DBL3-specific Abs from pregnant women with malaria. NK cells were assessed for surface CD107a expression and intracellular IFNγ and TNFα production in the presence of VAR2CSA subdomains DBL2 (a–c) or DBL3 (d–f). Percentage of activation markers expressed by NK cells (mean of three separate donors) are shown. NK cells were stimulated with purified IgG Abs from pregnant women mid pregnancy with placental malaria (PM; N = 50; red) or from pregnant women with non-placental malaria (NP; N = 27; blue) at delivery in the presence of VAR2CSA subdomains DBL2 or DBL3. IgG Abs from malaria-naïve donors were used as negative control (N = 8; grey). Activation marker expression of NK cells incubated without Abs and median of SIV gp120-specific responses were subtracted as background. Statistical comparison between groups was performed using a Kruskal–Wallis test corrected for multiple comparisons using Dunn’s multiple comparison method (p-values are shown on graphs).

Figure 4

Polyfunctional responses of NK cells induced by DBL2-specific Abs from pregnant women with malaria. NK cells from three separate donors were stimulated with IgG from pregnant women mid pregnancy with non-placental malaria (N = 27), women with placental malaria (N = 50) at delivery in presence of DBL2 and assessed for expression of CD107a, IFNγ and TNFα. (a) NK cells were selected based on their CD56 expression (CD56^dim and CD56^bright). Pie and Bar charts show the proportion (b) and relative frequency (c) of each activation marker combination of only activated NK cells. (b) The pie segments correspond to NK cells expressing different combinations of activation markers and are color coded (pie segment legend: pink-red) to indicate increasing polyfunctional NK cell activation. (c) The bar graph shows relative frequencies of combinations of activation markers by NK cells stimulated with IgG from non-placental malaria-infected women (blue) or women with placental malaria (red) in presence of DBL2. Mean of three NK cell donors with standard deviation is shown. Statistical analysis between groups was performed using multiple t tests corrected for multiple comparisons using the Holm-Šídák method.

Figure 5

Polyfunctional responses of NK cells induced by DBL3-specific Abs from pregnant women with malaria. NK cells from three separate donors were stimulated with IgG from pregnant women mid pregnancy with non-placental malaria (N = 27), women with placental malaria (N = 50) at delivery in presence of DBL2 and assessed for expression of CD107a, IFNγ and TNFα. NK cells were selected based on their CD56 expression (CD56^dim and CD56^bright). Pie and Bar charts show the proportion (a) and relative frequency (b) of each activation marker combination of only activated NK cells. (a) The pie segments correspond to NK cells expressing different combinations of activation markers and are color coded (pie segment legend: pink-red) to indicate increasing polyfunctional NK cell activation. (b) The bar graph shows relative frequencies of combinations of activation markers by NK cells stimulated with IgG from non-placental malaria-infected women (blue) or women with placental malaria (red) in presence of DBL3 (bottom bar graph). Mean of three NK cell donors with standard deviation is shown. Statistical analysis between groups was performed using multiple t tests corrected for multiple comparisons using the Holm-Šídák method.

Figure 6

Glycan profiles of IgG Abs in pregnant women with malaria. (a) Schematic representation of N-linked glycan composition of human IgG Abs. The glycans are attached to asparagine (N) at position 297 in the C_H2 domain of IgG and have a biantennary heptasaccharide core (solid line) and variable extensions (dash line), such as fucose, galactose and/or sialic acid. Relative abundance of specific types of N-linked glycan structures of purified IgG Abs from non-infected pregnant women (NIP; N = 41; blue), pregnant women with malaria infection (IP; N = 11; pink), malaria-naïve healthy pregnant women (HP; N = 10; yellow) and uninfected healthy non-pregnant women (H; N = 13; grey) were profiled. % of Fc glycans with the presence of (b) galactose (monogalactosylated or digalactosylated), (c) sialic acid and (d) fucose. (e–l) The relative prevalence of several major glycan structures (G0 agalactosylated, G1 monogalactosylated, G2 digalactosylated, F fucosylated, S1 sialylated). Statistical comparison between groups was performed using a Kruskal–Wallis test corrected for multiple comparisons using Dunn’s multiple comparison method (p-values are shown on graphs).