Figure 1.
Characterization and longitudinal analysis of MDSC populations in cord blood, infants and adults.
(A) Gating strategy and identification of HLA-DR/CD14neg, CD33/CD11b/CD15pos G-MDSC in adult and cord blood. Further characterization of HLA-DR/CD14neg, CD33/CD11b/CD15pos cells by intracellular staining of Arginase I. (B) Frequency of HLA-DR/CD14neg, CD33/CD11b/CD15pos 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. (C) Wright-Giemsa cytospin of CB samples and phenotype determination by clinical pathology of neutrophils and G-MDSC (Average, n = 2 independent experiments). Magnification 600X. (D) Proportions of neutrophils at various stages of development in the neutrophil and the G-MDSC fractions.
Figure 2.
Effect of neonatal G-MDSC on T cell proliferation.
(A) 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) 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. (C) Suppression of T cell proliferation by autologous G-MDSC titration. (n = 4 independent experiments performed in duplicate). (D) Suppression of T cell proliferative responses by G-MDSC is contact dependent. (n = 5 independent experiments performed in duplicate). (E) 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).
Figure 3.
Effect of G-MDSC on IFN-gamma production.
(A) Cord blood CD3pos T cells and adult CD3pos CD45ROneg 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) Neonatal G-MDSC decrease IFN-gamma production after anti-CD3/CD28 bead stimulation. (n = 13 independent experiments performed in triplicate). (C) 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).