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The number of antigen-specific naive CD8+T cells is believed to be important in the shaping of adaptive immune responses, and is predictive for the magnitude of priming responses in mouse models. Because of extremely low precursor frequencies, knowledge about these cells comes from indirect techniques and estimations. Here, we present a strategy based on the combination of tetramer staining, magnetic-bead enrichment, and multiparametric cytometry, which permitted direct detection and analysis of CD8+T cells reactive for 6 different naive epitopes (MART-126-35, HIV-1 Gag p1777-85, hepatitis C virus [HCV] NS31406-1415, HCV Core132-140, NY-ESO-1157-165, and cytomegalovirus [CMV] pp65495-503). Interestingly, we detected higher than 100-fold differences in precursor frequency across these epitopes (from 0.6 × 10−6to 1.3 × 10−4), but conserved frequencies among humans. Development of a procedure for direct assessment of T-cell precursor frequency in humans has important implications, with particular relevance to vaccine development and monitoring of tumor and self-reactive T cells.

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis in young children. Microbial agents such as endotoxin and RSV are implicated in airway inflammation during the development of reactive airway disease (RAD) later in childhood. Toll-like receptors (TLRs) are involved in an inflammation cascade through pathogen-associated molecular pattern recognition including lipopolysaccharide (LPS) and viral components. In this study, we investigated the expression of TLRs and cytokine-chemokine production profiles of RSV-infected epithelial cells. In live-RSV infected human tracheal epithelial cell line (9HTEo), TLRs 1–10 mRNA levels were up-regulated in a time-dependent manner compared with ultraviolet (UV)-inactivated RSV. RSV was shown to alter TLR4 membrane and cytosolic location in epithelial cells. Stimulating RSV-infected epithelial cells with TLR4 agonist LPS increased synthesis of IL-6, IL-8, and reduced regulated on activation, normal T cell expressed and secreted (RANTES) production. TLR4 neutralizing antibody HTA125 and TLR4-targeting RNA interference experiments revealed that TLR4 signaling pathway played a predominant role in mediating LPS-induced-IL-6 production of RSV infected epithelial cells. Altogether, our studies indicated that TLR4 play a critical role in leading LPS mediated-IL-6 response in RSV infected-epithelial cells and might be an important factor influencing the cytokine-chemokine profile of epithelial cells interacting with virus and endotoxin, which is correlated with phenotypes of RSV diseases.

IGF-I has profound effects on the immune system. We previously reported that IGF-I promoted cord blood (CB)-naïve T-cell maturation and now show that IGF-I promoted maturation of CB monocyte–derived dendritic cells (DC) with up-regulation of CD83, CD86, CD40, and major histocompatibility complex (MHC) class II molecules, and down-regulation of mannose receptor. Furthermore, IGF-I inhibited apoptosis of CB DC and increased the production of tumor necrosis factor (TNF-). These effects were blocked by specific mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059) and phosphoinositol 3-kinase inhibitor (LY294002). PD98059 partially inhibited the IGF-I–induced up-regulation of MHC class II. In contrast, LY294002 was additive in the IGF-I–induced up-regulation of MHC class II. Moreover, LY294002 significantly increased the percentage of late apoptotic cells in CB. These results imply the involvement of different pathways for the differential regulation of co-stimulatory molecule expression and apoptosis. The addition of anti–TNF- did not neutralize the effects of IGF-I on CB DC maturation and apoptosis. On the contrary, neutralizing TNF- significantly increased the IGF-I–induced up-regulation of CD83 and CD40. We conclude that IGF-I has maturation and survival effects on CB DC. These effects are mediated through both MEK and PI 3-kinase pathways but not through the IGF-I induction of TNF- production by the DC.

Mycobacterium bovisbacillus CalmetteGuerin BCG is given to millions of neonates in developing countries as a vaccine against Mycobacterium tuberculosishowever, little is known about the initiation of response in neonatal dendritic cells DCs to BCG. To address this issue, the interaction of BCG with human cord blood monocytederived DCs was studied. We showed that BCG could promote cord blood monocytederived DC maturation by upregulation of CD80, CD83, CD86, CD40, and MHC class II molecules and downregulation of mannose receptor. BCG was able to induce similar levels of tumor necrosis factor and IL10 but no bioactive IL12p70 production from cord blood DCs as from adult blood DCs. Functionally BCGtreated cord blood DCs had higher ability to induce mixed lymphocyte reaction than nonBCGtreated cord blood DCs. Both nonBCGtreated and BCGtreated cord blood DCs efficiently induced a high level of IL10, medium level of interferon, but little IL4 production by cord blood naïve CD4T cells. Heat shock protein 65, a key component of BCG, had no effect on cord blood DC maturation in terms of CD86, MHC class II, and mannose receptor upregulation. During the BCGinduced maturation process of cord blood DCs, nuclear transcription factor RelB was upregulated and cytosolic RelB downregulated with cytosolic IB and degradation. These results suggest that BCG can promote cord blood monocytederived DC maturation, and that the mechanism is through the upregulation of nuclear RelB secondary to the degradation of cytosolic IB and . Pediatr Res54 105112, 2003

Neonates are relatively immature in their immune response; thus, to further clarify the differences of monocyte function and differentiation between neonates and adults, we investigated their CD14CD4and CD14CD16monocyte subpopulations, production of IL-1 and tumor necrosis factor- induced by lipopolysaccharide, and their CD14 and CD1a phenotypic changes in response to IL-4 and granulocyte-macrophage colony-stimulating factor. Our results showed that 1) the expression of CD14 in cord blood monocytes was significantly lower than that in adult peripheral blood monocytes;2) both the percentages of CD14CD4cells and CD14CD16cells among CD14monocytes were also significantly lower in cord blood;3) after stimulation by lipopolysaccharide for 72 h, production of both IL-1 and tumor necrosis factor- was lower in cord blood than that in adult peripheral blood; and 4) in response to IL-4 or GM-CSF, the phenotype development of CD14 and CD1a in cord blood and adult peripheral blood was different. Down-regulation of CD14 expression in response to IL-4 and GM-CSF was slower in cord blood monocytes than that in adult peripheral blood monocytes. After 9 d of culture in the presence of IL-4 and GM-CSF, the percentage of CD1amonocytes was significantly more increased in cord blood than that in adult peripheral blood. The reduced expression of CD14 and other mature phenotype markers such as CD16 and CD4 as well as the reduced IL-1 and tumor necrosis factor- production may contribute to the impaired immune response of neonates. Slower down-regulation of CD14 by IL-4 and GM-CSF suggests that differential properties of cord blood monocytes in response to cellular stress signals take a longer time than those of adult peripheral blood monocytes.