The earliest lymphoid precursor population in the adult mouse thymus had previously been shown to produce not only T cells, but also dendritic cell (DC) progeny on transfer to irradiated recipients. In this study, culture of these isolated thymic precursors with a mixture of cytokines induced them to proliferate and to differentiate to DC, but not to T lineage cells. At least 70% of the individual precursors had the capacity to form DC. The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures. The cultured DC also expressed high levels of class I and class II major histocompatibility complex, together with CD11c, DEC-205, CD80, and CD86, markers characteristic of mature DC in general. However, they did not express CD8α or BP-1, markers characteristic of normal thymic DC. The optimized mixture of five to seven cytokines required for DC development from these thymic precursors did not include granulocyte/macrophage colony stimulating factor (GM-CSF), usually required for DC development in culture. The addition of anti–GM-CSF antibody or the use of precursors from GM-CSF–deficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) 3 and IL-7 were present, although some stimulation by GM-CSF was noted in their absence. In contrast, DC development was enhanced by addition of the Flt3/Flk2 ligand, in line with the effects of the administration of this cytokine in vivo. The results indicate that the development of a particular lineage of DC, probably those of lymphoid precursor origin, may be independent of the myeloid hormone GM-CSF.

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Cytomegalovirus (CMV) infection is endemic in Gambian infants, with 62% infected by 3 months and 85%by 12 months of age. We studied the CD8 T-cell responses of infants to CMV following primary infection. CMV-specific CD8 T cells, identified with tetramers, showed a fully differentiated phenotype (CD28. Strikingly, the overall CD8 T-cell population developed a similar phenotype following CMV infection, which persisted for at least 12 months. In contrast, primary infection was accompanied by up-regulation of markers of activation (CD45R0 and HLA-D) on both CMV-specific cells and the overall CD8 T-cell population and division (Ki-67) of specific cells, but neither pattern persisted. At 12 months of age, the CD8 T-cell population of CMV-infected infants was more differentiated than that of uninfected infants. Although the subpopulation of CMV-specific cells remained constant, the CMV peptidespecific gamma interferon response was lower in younger infants and increased with age. As the CD8 T-cell phenotype induced by CMV is indicative of immune dysfunction in the elderly, the existence of a similar phenotype in large numbers of Gambian infants raises the question of whether CMV induces a similarly deleterious effect.

The induction of dendritic cell (DC) maturation is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. In this study, we have investigated the effects of monophosphoryl lipid A (MPL) on human monocyte-derived DC as well as peripheral blood T cells. Calcium mobilization, mitogen-activated protein kinase activation, and the NF-κB transcription factor were induced after MPL stimulation of DC and required high doses of MPL (100 μg/ml). Maturation parameters such as production of IL-12 and increases in cell surface expression of HLA-DR, CD80, CD86, CD40, and CD83 were observed following DC treatment with MPL. However, lower levels of IL-12 were induced by MPL when compared with lipopolysaccharide. This is likely to be related to differences in the kinetics of extracellular signal-related kinase 1/2 and p-38 phosphorylation induced by both molecules. Although maturation induced by MPL was weaker when compared with lipopolysaccharide, it appeared to be sufficient to support optimal activation of allogeneic naive CD45RA+ T cell and anti-tetanus toxoid CD4 T cells. MPL at low doses (5 μg/ml) had no impact on DC maturation, while its addition to DC-T cell cocultures induced full T cell activation. The observed effect was related to the fact that MPL also acts directly on T cells, likely through their Toll-like receptors, by increasing their intracellular calcium and up-regulating their CD40 ligand expression. Together, these data support a model where MPL enhances T cell responses by having an impact on DC and T cells.

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SUMMARYThe effects of exposure to placental malaria infection on newborn immunological responses, in particular Th1/Th2 cytokines and antigen-presenting cell (APC) function, were compared between cord blood mononuclear cells (CBMC) from parasitized and non-parasitized placentas of Gambian women. Cells were analysed in vitro for their ability to respond to mitogens [phorbol myristate acetate (PMA)/ ionomycin, phytohaemagglutinin (PHA)], a malaria-unrelated test antigen [purified protein derivative of Mycobacterium tuberculin [purified protein derivative (PPD)] and Plasmodium falciparum schizont extracts. Mitogens induced strong proliferation and secretion of high concentrations of both IL-13 and sCD30 in CBMC from both groups. Conversely, significantly lower amounts of IFN-g were induced in the parasitized group in response to low doses of PHA. Protein antigens induced very low amounts of all tested cytokines, in particular IFN-g . However, a significantly higher release of sCD30 was observed in response to schizont extracts in the parasitized group. Addition of LPS to activate APC to low doses of PHA or schizont extracts increased the IFN-g production in both groups but levels remained lower in CBMC from the parasitized group. This result correlates with the lower production of IL-12 found following lipopolysaccharide (LPS) stimulation in this group. Taken together, these data show that placental infection with P. falciparum affects Th1 differentiation and sCD30 priming of neonatal lymphocytes and that the probable mode of action is via APC.

BackgroundCongenital cytomegalovirus (CMV) infection is the most prevalent congenital infection worldwide. Epidemiology and clinical outcomes are known to vary with socio-economic background, but few data are available from developing countries, where the overall burden of infectious diseases is frequently high.Methodology/Principal FindingsAs part of an ongoing birth cohort study in The Gambia among term infants, urine samples were collected at birth and tested by PCR for the presence of CMV DNA. Risk factors for transmission and clinical outcome were assessed, including placental malaria infection. Babies were followed up at home monthly for morbidity and anthropometry, and at one year of age a clinical evaluation was performed. The prevalence of congenital CMV infection was 5.4% (40/741). A higher prevalence of hepatomegaly was the only significant clinical difference at birth. Congenitally infected children were more often first born babies (adjusted odds ratio (OR) 5.3, 95% confidence interval (CI) 2.0-13.7), more frequently born in crowded compounds (adjusted OR 2.9, 95%CI 1.0-8.3) and active placental malaria was more prevalent (adjusted OR 2.9, 95%CI 1.0-8.4). These associations were corrected for maternal age, bed net use and season of birth. During the first year of follow up, mothers of congenitally infected children reported more health complaints for their child.Conclusions/SignificanceIn this study, the prevalence of congenital CMV among healthy neonates was much higher than previously reported in industrialised countries, and was associated with active placental malaria infection. There were no obvious clinical implications during the first year of life. The effect of early life CMV on the developing infant in the Gambia could be mitigated by environmental factors, such as the high burden of other infections.

SummaryPlacental malaria infection affects the T helper type 1 (Th1)/Th2 balance in neonatal children. We investigated a potential role of regulatory T cells in this balance by comparing T cell responses of cord blood mononuclear cells (CBMC) from parasitized and non-parasitized placenta of Gambian women. CBMC were depleted of CD4 + CD25 + forkhead box P3 (FoxP3) + regulatory T cells and analysed in vitro for their ability to produce interferon (IFN)-g, sCD30 and interleukin (IL)-10 in response to phytohaemagglutinin (PHA), live Plasmodium falciparum, schizont extracts and the recombinant P. falciparum blood stage antigen merozoite surface protein 1 (MSP119). As expected, lower IFN-g and higher sCD30 responses were observed for the cells from the parasitized group. In addition, higher IL-10 levels were produced by CBMC from the parasitized group. Depletion of regulatory T cells decreased IL-10 production, which resulted in a restoration of IFN-g expression in response to all stimuli. The Th2 marker sCD30 remained significantly higher in the parasitized group in response to malaria protein antigens while similar levels were recovered between both groups in response to live P. falciparum. Similar effects were observed by adding an antibody that blocks IL-10 function. These results suggest that the impact of P. falciparum infection on Th1 differentiation of neonatal T cells can be ascribed to regulatory T cells through production of IL-10.