Human NK Cell Up-regulation of CD69, HLA-DR, Interferon γ Secretion and Cytotoxic Activity by Plasmacytoid Dendritic Cells is Regulated through Overlapping but Different Pathways.

Human plasmacytoid dendritic cells secrete high levels of IFNα and are thus implicated in the activation of NK cells. Activated NK cells are characterised by the up-regulation of CD69 and MHC class II DR expression, secretion of IFN γ and enhanced cytotoxicity. We show that pDC mediate these processes by different mechanisms, some of which overlap. Human NK cells were analysed after co-culture with immature or CpG-matured blood pDC or with supernatant from these cells. Maximal CD69 expression by NK cells was mediated by supernatant from mature pDC and did not require pDC contact. Up-regulation was due in part to IFNα but also to factors in IFNα negative supernatant from immature DC. HLA-DR expression was independent of secreted molecules but required contact with immature or mature DC. Enhanced NK cytotoxicity, measured by killing of K562 targets and expression of CD107a, was mediated by multiple factors including type I IFN, supernatant from immature pDC cultures and contact with immature or mature pDC. These factors act cumulatively to enhance cytotoxcity. Thus different parameters of pDC mediated NK cell activation are regulated by distinct pathways.

Human Langerhans' cells and dermal-type dendritic cells generated from CD34 stem cells express different toll-like receptors and secrete different cytokines in response to toll-like receptor ligands.

Langerhans' cells (LC) and dermal dendritic cells (dDC) are located in the superficial and deeper layers of the skin respectively and represent the main dendritic cell (DC) populations of the skin. LC-like and dDC-like DC can be generated from CD34 stem cells and this system is widely used as a model for investigating these cells and in therapeutic vaccination. Here we report toll-like receptor (TLR) expression in human LC and dDC derived from CD34 stem cells. In vitro-generated DC expressed TLR-1, 2, 4, 6, 8 and 10. LC, but not dDC, expressed TLR-5, whereas only dDC expressed TLR-3. Maturation of LC was mediated by TLR-2, 4 and 5 ligands, but not by a TLR-3 ligand. dDC maturation was induced by TLR-3 and -4, but not with TLR-5 ligand and only weakly by a TLR-2 ligand. Stimulated LC secreted interleukin (IL)-1beta, low levels of tumour necrosis factor-alpha (TNF-alpha) and IL-8, but not IL-6 or IL-10. dDC secreted TNF-alpha, IL-6, IL-8 and IL-10, but little IL-1beta. IL-12p70 was not produced by ligand-stimulated dDC or LC, but was secreted by monocyte-derived DC (mdDC) stimulated with lipopolysaccharide (LPS). Thus, in vitro-generated LC and dDC detect different pathogen-associated molecules and show different cytokine-secretion profiles in response to TLR ligands.

Activity of different vaccine-associated promoter elements in human dendritic cells.

Vaccine design approaches that target dendritic cells (DC) aim at achieving high levels of transgene expression. Careful selection of the promoter element driving the foreign gene is therefore important. We have constructed adenovirus vectors carrying the gene for enhanced green fluorescent protein (eGFP) driven by three different promoters, CMV, CMV5 and ubiquitin C (UbC) promoter, and analysed their activity in different populations of human DC, namely blood plasmacytoid (pDC) and myeloid DC (mDC), monocyte-derived DC (moDC), Langerhans (LC) and dermal type DC (dDC). Although the CMV5 promoter was more active than the other two promoters in the HeLa and 911HER cell lines, in human DC the highest level of transgene expression was seen with the CMV promoter. There was very low-level eGFP expression in all cell types transduced with the UbC promoter. Highest eGFP expression levels were observed in moDC, cultured mDC and LC and the lowest levels in pDC. Expression of eGFP was augmented in all DC populations upon stimulation with CD40 ligand (CD40L). These findings demonstrate that the CMV promoter is the most effective of the three promoters tested in a range of different human DC populations.

Human blood CD1c dendritic cells stimulate IL-12-independent IFN-γ responses and have a strikingly low inflammatory profile.

Adaptive immune responses are initiated by resident myeloid tissue DC. A major fraction of tissue DC express CD1c+ and is thought to be derived from blood CD1c DC, an idea supported here by the observation that they express tissue-homing molecules and rapidly differentiate into cells with a tissue DC phenotype. Responses are thought to be augmented/modulated further by inflammatory moDC. Although much accepted human myeloid DC cell biology is based on moDC studies, we find these 2 DC populations to be functionally distinct. Stimulated moDC produce high levels of IL-10 and the Th1-promoting cytokine IL-12. Under identical conditions, CD1c DC synthesized no IL-10 and no or low levels of IL-12. Despite this, CD1c DC stimulated a strong Th1 response, demonstrated by IL-12 neutralization to be IL-12 independent, whereas the response induced by moDC was IL-12 dependent. This finding was supported by studies on a patient with a highly reduced ability to synthesize IL-12, whose CD1c DC induced a good Th1 response contrasting with the failure of his moDC, which were impaired in IL-12 production, to induce IFN-γ-secreting T cells. The IL-10 and IL-12 data were confirmed by microarray analysis, which also showed that stimulated moDC produced inflammatory-associated chemokines and cytokines, whereas stimulated CD1c DC showed minimal up-regulation of these genes. Thus, moDC, widely used as a human myeloid DC model, do not faithfully reflect the properties of CD1c tissue DC, making the initial response to a pathogen or vaccine.

Synchronous sperm retrieval and sperm washing in an intracytoplasmic sperm injection cycle in an azoospermic man who was positive for human immunodeficiency virus.

OBJECTIVE: To present the first reported case of synchronous sperm retrieval followed by sperm washing before an intracytoplasmic sperm injection (ICSI) cycle in an HIV-positive azoospermic man. DESIGN: Case report. SETTING: Assisted reproduction center. PATIENT(S): A 40-year-old HIV-positive man with obstructive azoospermia due to vasal aplasia. INTERVENTION(S): Synchronous sperm retrieval, sperm washing, nucleic acid-based sequence amplification testing, and intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Successful sperm retrieval sufficient for sperm washing and fertilization. RESULT(S): Sufficient quantity of spermatozoa for washing was obtained at epididymal aspiration. After the wash, HIV ribonucleic acid (RNA) was undetectable with nucleic acid-based sequence amplification testing, enabling injection of oocytes collected after routine gonadotropin superovulation. Of seven oocytes collected from the 39-year-old woman partner, six were injected and five fertilized (83%). Three embryos were transferred on day 2. The pregnancy test was negative on this occasion. CONCLUSION(S): This case demonstrates that sperm washing can be applied in cases of sperm retrieval where sperm volume and density is low, allowing the treatment of azoospermic HIV-positive men.

Langerhans cells are more efficiently transduced than dermal dendritic cells by adenovirus vectors expressing either group C or group B fibre protein: implications for mucosal vaccines.

Vaccines against viruses need to target dendritic cells (DC) and stimulate mucosal immunity. Most vaccine studies have focussed on monocyte-derived or dermal DC (dDC) but recent evidence suggests that Langerhans cells (LC) may stimulate mucosal immunity more effectively. New chimeric adenovirus vectors expressing fibre protein from group B adenoviruses (rAd5/11), which utilise CD46 rather than the Coxsackie adenovirus receptor (CAR), have been developed as vaccines to improve transduction and overcome problems of pre-existing vector immunity. Transduction of LC and dDC by rAd5/11 and standard rAd5 expressing green fluorescent protein (GFP) showed that both DC types were more efficiently transduced by rAd5/11 than by rAd5. Although expression of CD46 and the integrins alphavbeta3 and alphavbeta5, which recognise the adenovirus penton base and mediate virus internalisation, was similar in LC and dDC, LC expressed higher levels of GFP. Transduction by electroporation of plasmid also resulted in higher GFP expression in LC, suggesting differences between the two DC populations at a post-entry stage. Transduction with either vector did not induce maturation of LC or dDC and did not affect their ability to stimulate T cells. These findings suggest that vaccine strategies that target LC with adenovirus vectors may be worthy of exploration.