Table 1.
Characteristics of healthy donors and patients.
Fig 1.
Frequencies and characteristics of NK cells in patients infected with DENV-2, CHIKV, co-infected with CHIKV and DENV-2 (CHIKV/DENV-2), and non-infected and healthy Gabonese controls (NI).
Patients were sampled in early acute (EA; day (D)0-D3), late acute (LA; D12-D15) and convalescent (C; D>30) stages post-onset of symptoms. Frequencies of CD3-CD56+ NK cells within the CD45+ lymphocyte gate (A), frequencies of early activation marker CD69 (B) and NKp30, NKG2A, CD161, CD57 cell-surface receptors (C) on CD3-CD56+ NK cells are represented. * p<0.05, ** p<0.001.
Fig 2.
Persistence of NKG2C+CD57+ NK cells in CHIKV-infected patients with high viral loads.
Results are grouped according to 1) type of infection: DENV-2, CHIKV or co-infected by CHIKV and DENV-2 (CHIKV/DENV-2), and non-infected Gabonese healthy controls (NI) and 2) date of sample collection after onset of symptoms: early acute (EA; day (D)0-D3), late acute (LA; D12-D15) and convalescent (C; D>30). (A) Frequency of NKG2C+ cells within CD3-CD56+ NK cells amongst all patient groups (B) Frequency of CD57+ NK cells within NKG2C+ or NKG2C- NK cell-subsets from CHIKV-, DENV-2- and co-infected patients analyzed at Day 30 post-symptoms. (C) Scatter plot representation of CD57+ and NKG2C+ NK cell frequencies collected in CHIKV-infected patients. Dotted vertical lines represent the baseline levels. (D) Correlation between the set point of the viral load quantified during the early acute CHIKV infection and CD57+ or NKG2C+ NK cells analyzed at Day 30 post-symptoms. Dotted horizontal lines represent baseline levels. * p<0.05, ** p<0.001.
Fig 3.
Association of KIR2DL1 expression and DENV-2 infection.
Results are grouped according to type of infection: DENV-2, CHIKV or co-infected by CHIKV and DENV-2 (CHIKV/DENV-2), and compared to non-infected Gabonese healthy controls (NI). Samples from infected patients were collected in early acute (EA; day (D)0-D3), late acute (LA; D12-D15) and convalescent (C; D>30) stages post-onset of symptoms. Frequency of KIR2DL1 (A) and KIR2DL2/2DL3 (B) gated on CD3-CD56+ NK cells. Dot-plots show only cells from EA patients. * p<0.05, ** p<0.001.
Fig 4.
Hierarchical clustering analysis of 9 NK-cell markers from patients infected with DENV-2 (DENV; Green; n = 15), CHIKV (CHIK; Blue; n = 19), co-infected with CHIKV and DENV-2 (COIFN; Orange; n = 9), and non-infected healthy Gabonese controls (NI; Purple; N = 15).
Patients were sampled in early acute (EA; day (D)0-D3), late acute (LA; D12-D15) and convalescent (C; D>30) stages post-onset of symptoms. Hierarchical clustering analysis was performed with the Genesis program (software available at www.genome.tugraz.at), as previously described [14,25]. Each column is dedicated to a definite NK marker. The color of each square reflects the percentage of expression of the corresponding marker in each individual. The values measured for samples were color displayed and rank ordered considering the healthy donors’ median as a reference: green color indicates inferior to median, and red color indicates superior to median, with values ranging from -3 to +3.
Fig 5.
Principal component analysis graphically showing the (A) statistical distribution of the individuals, according to the differential expression of NK-cell markers, and (B) statistical proximity between these different markers. DENV-2+ patients are noted in green, CHIKV+ patients in blue, and healthy donors in purple.
Fig 6.
Cytolytic markers and functional activity of NK cells in patients infected with DENV-2, CHIKV, co-infected with CHIKV and DENV-2 (CHIKV/DENV-2), and non-infected and healthy Gabonese controls (NI).
Patients were tested at early acute (EA; day (D)0-D3) stage post-onset of symptoms. (A) Intracellular staining of perforin and granzyme-B cytolytic markers on non-activated CD3-CD56+ NK cells. (B) CD107a expression on CD3-CD56+ NK cells stimulated by K562 target cells. Results are shown for an effector/target (E/T) cell ratio of 1/1. (C) Intracellular staining of IFN-γ+ in CD3-CD56+ NK cells after IL-12 plus IL-18 overnight stimulation. *: p<0.05; ***: p<0.0001.