Impaired Allogeneic Activation and T-helper 1 Differentiation of Human Cord Blood Naive CD4 T Cells

Reduced activation of CB naive CD4 T cells by allogeneic DCs. CD69 (A) and CD154 (B) expression on APB and CB naive CD4 T cells after incubation with or without allogeneic DCs for 24 hours. Cells were analyzed by flow cytometric analysis for CD4, CD45RA, and CD154 or CD69. DC1, DC2, and DC3 represent DC lines derived from 3 unrelated healthy adult blood donors, respectively, and DC1+2+3 indicates pool of these 3 DC lines. Events shown are gated for CD45RA⁺ lymphocytes, with boxed numbers indicating percentage of positive cells. Results shown are representative of at least 6 independent experiments.

Reduced cytokine production by co-cultures of CB naive CD4 T cells with allogeneic DCs. A, IFN-γ, IL-2, IL-4, IL-10, and IL-12 content in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA) of naive CD4 T cells from APB (open bars) or CB (closed bars) after 48 hours of co-culture with allogeneic DCs. *P < .05 compared with adult CD4 T-cell co-cultures. B, Kinetics of IFN-γ and IL-2 accumulation in supernatants of ABP (solid line) or CB (dashed line) naive CD4 T-cell co-cultures with allogeneic DCs by ELISA. Results are representative of 3 independent experiments.

IFN-γ production by naive CD4 T cells after allogeneic DC stimulation is dependent on IL-12 and CD154. A, APB or CB naive CD4 T cells were incubated with allogeneic DCs for 48 hours in presence or absence of 10 μg/mL of neutralizing CD154 or IL-12 p70 mAb or isotype-matched control mAbs. Intracellular IFN-γ accumulation in APB (top) or CB (bottom) naive CD4 T cells is shown. Events shown are CD45RA^high lymphocytes, with boxed numbers indicating percentage and MFI of IFN-γ⁺ cells. B, IFN-γ or IL-12 levels in supernatants of co-cultures of CD4 T cells and DCs. AB or CB naive CD4⁺ T cells (3 × 10⁵) were incubated with allogeneic DCs (3 × 10⁴) in presence or absence of anti-CD154 antibody (5c8), or anti-IL-12 or their isotype control antibodies IgG2a, or IgG1, at 10μg/mL. After 48 hours of culture, supernatants were harvested and measured for IFN-γ or IL-12 content by enzyme-linked immunosorbent assay. *P < .05 compared with isotype control mAb incubation. Results shown are representative of 3 independent experiments.

Reduced STAT4 expression and IL-12-induced tyrosine phosphorylation of STAT4 by CB naive CD4 T cells. A, STAT4 mRNA levels in CB and APB naive CD4 T cells immediately after isolation or in naive CD4 T cells expressing CD69 after 24 hours of alloantigenic stimulation. Transcript level for freshly isolated CB naive CD4 T cells was arbitrarily assigned value of 1.0 for comparison. Ct values were normalized using 18S ribosomal RNA as internal control. *P < .05 compared with corresponding CB CD4 T-cell subset. B, Intracellular STAT4 protein in APB (top) or CB naive (bottom) CD4 T cells, with number in left upper corner indicating percentage of cells staining positive by flow cytometry. C, Flow cytometric analysis of intracellular pY-STAT4 expression by allogeneically primed CB and APB naive CD4 T cells after brief treatment with IL-12. Events shown are for CD45RA⁺ CD4 T cells, with numbers indicating percentage of positive cells. For all panels, one of 3 independent experiments with similar results is shown.

Decreased FoxP3 expression by naive CD4 T cells of the CD25^high subset from CB compared with APB. Flow cytometric analysis of naive (CD45RA^high) CD4 T cells from APB (top) and CB (bottom) are shown. A, CD4 versus CD25 surface expression, with percentage CD25^−/low cells (low right quadrant) and percentage of CD25^high cells and MFI for CD25 of CD25^high population (upper right quadrant) indicated. B, Intracellular FoxP3 expression in freshly isolated CD25^high naive CD4 T cells, with percentage of positive cells indicated. Filled histogram (isotype control) is compared with solid line (FoxP3). C, Intracellular FoxP3 expression in freshly isolated CD25^−/low naive CD4 T cells. Filled histogram (isotype control) is compared with solid line (FoxP3), and percentage of positive cells is indicated.