Table 1.
Clinical characteristics of study participants.
Fig 1.
(A) Representative gating strategy employed for the identification of total NK cells and CD8+ T cells from lymphocytes using multi-parametric flow cytometry. Side scatter area (SSC-A) vs forward scatter area (FSC-A) was used to identify lymphocytes followed by exclusion of doublets by forward scatter height (FSC-H) vs FSC-A, and exclusion of non-viable cells, B-cells and monocytes by use of viability dye and anti-CD19 and anti-CD14 monoclonal antibodies. Cytolytic T cells were identified using anti-CD3 and anti-CD8 monoclonal antibodies, whilst NK cells were identified as CD3 negative and CD56 positive and/or CD16 positive cells. (B) NK cell frequency as a percentage of total viable lymphocytes in all participants. NK cell frequency as a percentage of total viable lymphocytes stratified by DARC genotype in HIV negative (C) and HIV positive (D) individuals. Correlations between NK cells as a percentage of total lymphocytes and ANCs are shown in HIV negative (E) and HIV positive (F) individuals. Dots indicate individual data points. Medians are indicated and extended to interquartile range with whiskers. Abbreviations: DARC, Duffy antigen receptor for chemokines; ANC, Absolute neutrophil count; r, Spearman rho.
Fig 2.
NK cell subset frequencies and markers of differentiation.
(A) Total NK cells grouped by relative CD56 and CD16 expression into CD56 bright, CD56 dim and CD56 negative subsets. (B) NK cell subset frequencies in all participants. NK cell subset frequencies stratified by DARC genotype in HIV negative (C) and HIV positive (D) individuals. (E) Gating strategy for CD57 expressing NK cells as a percentage of total NK cells. (F) CD57 expressing NK cells as a percentage of total NK cells in all participants. CD57 expressing NK cells as a percentage of total NK cells stratified by DARC genotype in HIV negative (G) and HIV positive (H) individuals. Correlations between CD57 expressing NK cells and ANCs are shown in HIV negative (I) and HIV positive (J) individuals. (K) Representative strategy employed for the identification of KIR and/or NKG2A expressing NK cells. (L) KIR/NKG2A subsets as a percentage of total NK cells in all participants. KIR/NKG2A subsets further stratified by DARC genotype in HIV negative (M) and HIV infected (N) individuals. Dots indicate individual data points. Medians are indicated and extended to interquartile range with whiskers. Abbreviations: DARC, Duffy antigen receptor for chemokines; K+N-, KIR+NKG2A- NK cell subset; K+N+, KIR+NKG2A+ NK cell subset; K-N+, KIR-NKG2A+ NK cell subset; K-N-, KIR-NKG2A- NK cell subset.
Fig 3.
NK cell activation, exhaustion and degranulation.
(A) Gating strategy of activation marker HLA-DR on total NK cells. (B) Expression of activation marker HLA-DR on total NK cells in all participants. Expression of activation marker HLA-DR grouped by DARC genotype on total NK cells in HIV negative (C) and HIV positive (D) individuals. (E) Gating strategy of exhaustion marker PD-1 on total NK cells. (F) Expression of exhaustion marker PD-1 on total NK cells in all participants. Expression of PD-1 marker grouped by DARC genotype on total NK cells in HIV negative (G) and HIV positive (H) individuals. (I) Gating strategy of degranulation marker CD107a on total NK cells. (J) Expression of degranulation marker CD107a on total NK cells in all participants. CD107a expression on total NK cells stratified by DARC genotype in HIV negative (K) and HIV positive (L) individuals. Dots indicate individual data points. Medians are indicated and extended to interquartile range with whiskers. Abbreviations: DARC, Duffy antigen receptor for chemokines.
Fig 4.
NK cell survival and proliferation measurement.
(A) Gating strategy of Annexin V and propidium iodide on total NK cells. NK cells that survived were identified as Annexin (Ann)-PI-. (B) Percentage of NK cell survival for unstimulated and K-562+cytokine stimulated cells in all participants. Participants were further stratified to show percentage of NK survival for unstimulated and K-562+cytokine stimulated cells in HIV negative (C) and HIV positive (D) individuals by DARC trait. (E) Gating strategy of CFSE labeling on total NK cells. (F) Percentage of NK cells divisions (CFSE low) in all participants. Participants were further stratified to show percentage of NK cells divided in HIV negative (G) and HIV positive (H) individuals by DARC trait. Dots indicate individual data points. Medians are indicated and extended to interquartile range with whiskers. Abbreviations: DARC, Duffy antigen receptor for chemokines; CFSE, carboxyfluorescein succinimidyl ester.