[Show abstract][Hide abstract] ABSTRACT: Newborns are highly susceptible to viral infections. We hypothesized that this susceptibility could be due to a dysregulated expression of innate virus-sensing receptors, i.e., TLR3, TLR7, TLR8, and TLR9 and the cytosolic receptors retinoic acid-inducible gene I, melanoma differentiation-associated protein 5, protein kinase R, and IFN-γ-inducible protein 16. Cord blood mononuclear cells (CBMCs) expressed mRNA for all these receptors except for TLR3. In peripheral blood mononuclear cells (PBMCs), TLR3 mRNA was preferentially expressed in cytotoxic cells, particularly CD56(dim) NK cells. Cord NK cells in contrast showed low TLR3 mRNA expression and lacked TLR3 protein expression. Cord NK cells did not produce IFN-γ in response to polyinosinic-polycytidylic acid [poly(I:C)], whereas strong IFN-γ production was observed in poly(I:C)-stimulated adult NK cells. Cord NK cells had poor cytotoxic function that was only marginally enhanced by exposure to the TLR3 ligand poly(I:C). Opposite to NK cells from adults, their cytotoxicity was not improved by herpes simplex virus (HSV) exposure and they were unable to kill HSV-infected cells. There were no differences in the TLR3 mRNA levels among men, women, and pregnant women, implying that TLR3 is not under sex hormone control. However, decidual NK cells expressed low levels of TLR3 mRNA, which was attributed to their CD56(bright) phenotype. Our data show that cord blood NK cells have deficient TLR3 expression associated with an inability to respond to poly(I:C) and HSV activation and to kill HSV-infected cells. This might explain why newborns are particularly sensitive to neonatal HSV infections.
[Show abstract][Hide abstract] ABSTRACT: Indoleamine 2,3-dioxygenase (IDO), a tryptophan-metabolizing enzyme expressed by dendritic cells (DC), has the potential to inhibit T cell responses and to promote tolerance. In contrast, cholera toxin (CT), the enterotoxin produced by Vibrio cholerae, promotes T cell responses, partly through its ability to induce DC maturation and promote antigen presentation. We hypothesized that the adjuvant activity of CT is associated with a lack of induction of IDO in DC. To test this hypothesis, monocyte-derived DC were pulsed with CT, and the IDO mRNA expression, IDO functional activity and cytokine production were measured as well as the ability of DC to induce T cell responses in vitro. Cholera toxin exposure induced enhanced levels of IDO mRNA in DC but no functional IDO protein activity. Cholera toxin pulsing however primed DC for CD40L-induced IDO protein activity. CD40L stimulation of CT-pulsed DC induced a modest IL-12p40 production, but not IL-12p70 or IL-23 secretion. Furthermore, CT-pulsed DC induced strong allogeneic and autologous T cell responses in vitro, which were not affected by the IDO-specific inhibitor 1-methyl tryptophan. Our results show that CT per se does not induce the expression of functional IDO protein, although it primes DC for CD40L-mediated IDO production and IL-12p40 secretion. Furthermore, CT-treated DC were equally powerful in their T cell stimulatory capacity as cytokine-matured DC.
[Show abstract][Hide abstract] ABSTRACT: We have evaluated whether cholera toxin (CT) as a carrier/adjuvant can enhance human T-cell responses to a viral oncoprotein in vitro using dendritic cells (DCs) as antigen-presenting cells. Monocyte-derived DCs obtained from women with cervical dysplasia were pulsed with the HPV16 oncoprotein E7, either alone or conjugated to CT, and tested for their ability to induce antigen-specific activation of autologous T cells in vitro. CT-conjugation of E7 significantly improved the capacity of pulsed DCs to activate antigen-specific CD4+ T-cell proliferation and IFN-gamma secretion. The CT-E7-pulsed DCs also produced significantly more of the Th1-inducing cytokine IL-12 compared to DCs pulsed with E7 or CT alone. Furthermore, DCs pulsed with CT-conjugated HPV16 E7 caused a response in T cells from women with advanced disease (CIN III) as well as in T cells from women that were currently not infected with HPV16. These data show the potential of using CT-conjugated viral oncoproteins for DC-induced T-cell activation in humans.