Healthy Neonates Possess a CD56-Negative NK Cell Population with Reduced Anti-Viral Activity

<div><p>Background</p><p>Neonatal Natural Killer (NK) cells show functional impairment and expansion of a CD56 negative population of uncertain significance.</p><p>Methods</p><p>NK cells were isolated from cord blood and from adult donors. NK subpopulations were identified as positive or negative for the expression of CD56 and characterized for expression of granzyme B and surface markers by multi-parameter flow cytometry. Cell function was assessed by viral suppression and cytokine production using autologous lymphocytes infected with HIV. Activating (NKp30, NKp46) and inhibitory (Siglec-7) markers in healthy infants and adults were compared with viremic HIV-infected adults.</p><p>Results</p><p>Cord blood contained increased frequencies of CD56 negative (CD56neg) NK cells with reduced expression of granzyme B and reduced production of IFNγ and the CC-class chemokines RANTES, MIP1α and MIP1β upon stimulation. Both CD56pos and CD56neg NK subpopulations showed impaired viral suppression in cord blood, with impairment most marked in the CD56neg subset. CD56neg NK cells from cord blood and HIV-infected adults shared decreased inhibitory and activating receptor expression when compared with CD56pos cells.</p><p>Conclusions</p><p>CD56neg NK cells are increased in number in normal infants and these effectors show reduced anti-viral activity. Like the expanded CD56neg population described in HIV-infected adults, these NK cells demonstrate functional impairments which may reflect inadequate development or activation.</p></div

NK Cell-Mediated Suppression of Viral Replication using Autologous HIV-Infected CD4 Lymphocytes.

<p>CD56posCD16pos (56pos) or CD56negCD16pos (56neg) NK cell populations from cord blood (CB) (n = 9) or healthy adult donors (AD) (n = 3, repeated in up to 5 experiments) were sorted and cultured with autologous CD4 T cells infected with HIV <i>in vitro</i>. Viral replication was assessed by production of HIV p24 antigen and suppression calculated by comparison with the quantity of p24 produced from control infected CD4 T cells without NK effectors. (A) Individual experiments were performed using NK cells at effector : target (E∶T) ratios of 5∶1 from single adult and cord blood samples. Individual adult donors are denoted by color. Generalized estimating equations were used with an exchangeable correlation matrix to account for repeat measures from the same subjects. (B) Representative experiment showing HIV suppression by NK cells at E∶T ratios of 10∶1, 5∶1 and 1∶1 using CD4 and NK cells from a single adult and a single cord blood donor. Data are shown for effectors from adult CD56pos, and cord blood CD56pos and CD56neg NK subpopulations. Histograms depict mean + SEM for sample replicates.</p

Cytokine Production by NK Cell Sub-Populations from Adults and Infants in Response to HIV-Infected Lymphocytes.

<p>CD56posCD16pos (56pos) or CD56negCD16pos (56neg) NK cell populations were isolated from cord blood (CB) (n = 4) and from healthy adult donors (AD) (56pos n = 4, 56neg n = 2). NK cell cytokine production in response to HIV-infected autologous CD4 T cells was assessed from supernatants on day 3 of the <i>in vitro</i> viral suppression assay using an 18-plex Luminex assay. Data are shown from 4 individual viral suppression experiments with assays performed in at least duplicate. On two occasions, insufficient cells were obtained from the adult CD56neg sample to allow analysis in replicate. Data are available therefore from four adult subjects for CD56pos effectors but from only two for AD CD56pos cells. Cytokine concentrations (pg/ml) are shown after subtracting ‘background’ values from control HIV-infected CD4 T cells incubated without NK effectors. Bars represent the median and interquartile range. Data for cytokine production were compared between groups using the Mann-Whitney test, * p<0.05. MIP  =  macrophage inflammatory protein; RANTES  =  regulated upon activation, normal T cell expressed and secreted.</p

Granzyme B Expression in NK Cell Subpopulations from Cord Blood and Healthy Adult Subjects.

<p>(A) Representative plots showing granzyme B against CD56 expression for NK cells from adult and cord blood donors. (B) Fluorescence Minus One (FMO) plots employed in generating the data displayed. (C) Proportion of cells positive for granzyme B (GzmB+) among NK cell subpopulations isolated from cord blood (CB) (n = 10) and adult peripheral blood (AD) (n = 10). NK cells were defined as CD56 positive (56pos) or negative (56neg) and stained for granzyme B with expression assessed by flow cytometry. (D) Levels of granzyme B expression as measured by mean fluorescence intensity (MFI) in cord blood and in adult NK cell subpopulations. NK cells were identified from mononuclear cells by selecting live singlets from the lymphocyte population, gated to exclude CD3 (T lymphocytes), CD14 (monocytes), CD19 (B lymphocytes) or CD33 (myeloid cells & precursors), and characterized by CD56 and CD16 surface expression. Box plots indicate median and interquartile range (box) with data limits (whiskers). Data comparisons were performed between groups using the Mann-Whitney test.</p

Expression of NK Receptors among NK Cell Subpopulations from Cord Blood, HIV-Infected and Healthy Adults.

<p>Surface NK markers were assessed by flow cytometry on CD56posCD16pos (56pos) and CD56negCD16pos (56neg) NK cell subpopulations from cord blood (CB) (n = 10), healthy adult HIV-seronegative individuals (AD) (n = 10) and viremic HIV-infected donors (HIV) (n = 10). A) Proportion of NK subsets staining positive for each receptor. B) Mean fluorescence intensity (MFI) for receptor expression among NK subpopulations positive for each surface receptor. Data between groups were compared using the Mann-Whitney test, * p<0.05, ** p<0.01. Bars represent mean ± SEM. Siglec  =  sialic acid binding immunoglobulin-like lectin. Representative plots showing receptor expression together with the Fluorescence Minus One (FMO) plots employed in these analyses are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067700#pone.0067700.s001" target="_blank">Figure S1</a>.</p

NK Cell Subpopulations in Cord Blood and Adult Peripheral Blood from Healthy Subjects.

<p>(A) Identification of NK cells from stained mononuclear cell populations was accomplished by selecting the live, singlet, lymphocyte population. Cells were gated to exclude expression of CD3 (T lymphocytes), CD14 (monocytes), CD19 (B lymphocytes) or CD33 (myeloid cells & precursors), and further characterized by CD56 and CD16 surface expression. (B) Proportions of major NK cell subpopulations present in umbilical cord blood (CB) (n = 10) and adult peripheral blood (AD) (n = 10). Box plots represent median and interquartile range (box) with data limits (whiskers). Data were compared between groups using the Mann-Whitney test. (C) Fluorescence Minus One (FMO) plots employed in generating the data displayed.</p

Effect of neonatal G-MDSC on T cell proliferation.

<p>(<b>A</b>) Proliferative responses of purified T cells in the presence or absence of G-MDSC after anti-CD3/CD28 bead stimulation (n = 28 independent experiments performed in duplicate for CD3 plots, n = 6 for CD4 and CD8 plots). Significance determined by the Wilcoxon Matched-Pair Signed Rank test. (<b>B</b>) Proliferative responses of purified adult naïve T cells (n = 9 independent experiments performed in duplicate) compared to cord blood T cells (n = 28 independent experiments performed in duplicate) after anti-CD3/CD28 bead stimulation. Statistical significance determined by the Mann Whitney test. (<b>C</b>) Suppression of T cell proliferation by autologous G-MDSC titration. (n = 4 independent experiments performed in duplicate). (<b>D</b>) Suppression of T cell proliferative responses by G-MDSC is contact dependent. (n = 5 independent experiments performed in duplicate). (<b>E</b>) G-MDSC frequency correlates with suppression of T cell proliferation by G-MDSC. G-MDSC frequencies were correlated to suppression of T cell proliferation by G-MDSC using the Spearman rank correlation test (n = 16 independent experiments).</p

Effect of G-MDSC on IFN-gamma production.

<p>(<b>A</b>) Cord blood CD3^pos T cells and adult CD3^pos CD45RO^neg T cells were assessed for IFN-gamma production by ELISpot after anti-CD3/CD28 bead stimulation (n = 11 independent experiments performed in triplicate for neonates and 9 independent experiments for adults). Statistical significance determined by the Mann Whitney test. (<b>B</b>) Neonatal G-MDSC decrease IFN-gamma production after anti-CD3/CD28 bead stimulation. (n = 13 independent experiments performed in triplicate). (<b>C</b>) G-MDSC frequency correlation with suppression T cell of IFN-gamma production by G-MDSC. G-MDSC frequencies were correlated to suppression using the Spearman rank correlation test (n = 11 independent experiments).</p

Characterization and longitudinal analysis of MDSC populations in cord blood, infants and adults.

<p>(<b>A</b>) Gating strategy and identification of HLA-DR/CD14^neg, CD33/CD11b/CD15^pos G-MDSC in adult and cord blood. Further characterization of HLA-DR/CD14^neg, CD33/CD11b/CD15^pos cells by intracellular staining of Arginase I. (<b>B</b>) Frequency of HLA-DR/CD14^neg, CD33/CD11b/CD15^pos cells of: (i) CBMC isolated from CB collected from healthy pregnancies in Seattle, WA (n = 25); (ii) PBMC isolated from neonates in Cape Town, South Africa at 6-weeks of age (n = 9); (iii) PBMC isolated from 6–24 month-old infants in Seattle, WA (n = 29); (iv) and PBMC isolated from healthy adults in Seattle, WA (n = 28). Statistical significance determined by the Mann Whitney test. (<b>C</b>) Wright-Giemsa cytospin of CB samples and phenotype determination by clinical pathology of neutrophils and G-MDSC (Average, n = 2 independent experiments). Magnification 600X. (<b>D</b>) Proportions of neutrophils at various stages of development in the neutrophil and the G-MDSC fractions.</p