Adel Benlahrech

Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1

In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing α4β7 integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-γ production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition.

Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8+ T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c+ dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c+ MHCIIhi CD8αneg epithelial cell adhesion molecule (EpCAMneg) CD11b+ langerin (Lang; CD207)neg DCs, but neither Langerhans cells nor Lang+ DCs were required for CD8+ T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8+ T-cell priming by live rAdHu5 MAs.

Monocyte‐Derived Dendritic Cells from HIV Type 1–Infected Individuals Show Reduced Ability to Stimulate T Cells and Have Altered Production of Interleukin (IL)–12 and IL‐10

Monocyte-derived dendritic cells (MDDCs) have been used in therapeutic vaccination for cancer. A small number of studies have employed a similar approach to vaccinate human immunodeficiency virus (HIV)-infected individuals. We have thus analyzed the functional properties of MDDCs generated from HIV-infected individuals who either are receiving highly active antiretroviral therapy or are therapy naive. There was no difference in the MDDC phenotype or efficiency of MDDC generation between HIV-infected individuals and healthy control subjects. Despite this, the MDDCs derived from both groups of infected individuals were severely impaired in their ability to stimulate the proliferation of allogeneic T cells. Furthermore, production of interferon-gamma was reduced in T cells stimulated by MDDCs. These functional changes may be at least partly explained by reduced interleukin-12 and increased interleukin-10 secretion on stimulation with lipopolysaccharide and CD40 ligand. Our findings suggest that MDDCs used in therapeutic vaccination of HIV-infected individuals may show reduced potency.

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)‐1β, low levels of tumour necrosis factor‐α (TNF‐α) and IL‐8, but not IL‐6 or IL‐10. dDC secreted TNF‐α, IL‐6, IL‐8 and IL‐10, but little IL‐1β. 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.

HIV-1 infection and induction of interferon alpha in plasmacytoid dendritic cells.

PURPOSE OF REVIEW Loss of blood plasmacytoid dendritic cell (pDC) in HIV-1 infection is thought to impact on adaptive immune responses whilst the virus also induces aberrant interferon alpha (IFN-α) production that may fuel chronic immune activation and drive disease progression. Recent attention has focussed on the pathway of HIV-induced IFN-α production by pDC and the new data are reviewed here together with the pathway leading to infection. RECENT FINDINGS Attachment to CD4 and chemokine co-receptors is essential for HIV-1 infection. Although CD4, but not co-receptor binding, is a major route for passage to endosomes and triggering of IFN-α secretion this may also occur by CD4-independent mechanisms involving other receptors. In contrast to other Toll-like receptor (TLR)-7 ligands and RNA viruses that stimulate pDC to secrete IFN-α for 2-3  h, HIV-1-stimulated pDC can give sustained IFN-α production for up to 48  h which may contribute to chronic immune activation. This may reflect retention of HIV in early endosomes which also seems to be associated with incomplete maturation induced by HIV. SUMMARY HIV-1-pDC interactions contribute to pathogenesis through depletion and aberrant IFN-α production. New data on the pathway of pDC HIV-stimulated IFN-α secretion may facilitate therapy to reduce chronic immune activation and slow disease progression.

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 αvβ3 and αvβ5, 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.

Loss of NK stimulatory capacity by plasmacytoid and monocyte-derived DC but not myeloid DC in HIV-1 infected patients.

Dendritic cells (DC) are potent inducers of natural killer (NK) cells. There are two distinct populations in blood, myeloid (mDC) and plasmacytoid (pDC) but they can also be generated In vitro from monocytes (mdDC). Although it is established that blood DC are lost in HIV-1 infection, the full impact of HIV-1 infection on DC-NK cell interactions remains elusive. We thus investigated the ability of pDC, mDC, and mdDC from viremic and anti-retroviral therapy-treated aviremic HIV-1+ patients to stimulate various NK cell functions. Stimulated pDC and mdDC from HIV-1+ patients showed reduced secretion of IFN-α and IL-12p70 respectively and their capacity to stimulate expression of CD25 and CD69, and IFN-γ secretion in NK cells was also reduced. pDC activation of NK cell degranulation in response to a tumour cell line was severely reduced in HIV-1+ patients but the ability of mDC to activate NK cells was not affected by HIV-1 infection, with the exception of HLA-DR induction. No differences were observed between viremic and aviremic patients indicating that anti-retroviral therapy had minimal effect on restoration on pDC and mdDC-mediated activation of NK cells. Results from this study provide further insight into HIV-1 mediated suppression of innate immune functions.

Dysregulated immunophenotypic attributes of plasmacytoid but not myeloid dendritic cells in HIV-1 infected individuals in the absence of highly active anti-retroviral therapy.

Dendritic cells (DC) in HIV‐1‐infected individuals are decreased and their dysfunction has been implicated in HIV‐1 immunopathogenesis. The mechanism of their dysfunction remains unclear, thus we analysed the expression of membrane molecules associated with immune regulation and DC activation in myeloid (mDC) and plasmacytoid DC (pDC) in therapy‐naive and highly active anti‐retroviral therapy (HAART)‐treated HIV‐1+ patients. DC from healthy controls, untreated HIV‐1+ and HAART‐treated patients were assessed by flow cytometry for expression of: anergy and apoptosis inducing molecules [programmed death (PD)‐1 and its ligands PD‐L1 and PD‐L2], inhibitory and regulatory T cell‐inducing molecules [immunoglobulin‐like transcript (ILT)‐3 and ILT‐4], interferon (IFN)‐α inhibitory receptor (ILT‐7) and co‐stimulatory molecules (CD80, CD83, and CD86). pDC from untreated HIV‐1+ patients expressed significantly lower levels of ILT‐7 compared to healthy controls, while HAART‐treated patients showed normal expression. pDC were also found to express moderately higher levels of PD‐L1 and ILT‐3 and lower levels of PD‐L2 receptors in untreated patients compared to controls and HAART‐treated patients. No significant changes were observed in mDC. There were no associations between the percentages and levels of expression of these molecules by pDC and viral load or CD4 T cell count. In conclusion, pDC but not mDC from HIV‐1+ patients with active viraemia display higher levels of apoptosis and T regulatory‐inducing molecules and may be predisposed to chronically produce IFN‐α through down‐regulation of ILT‐7. HAART restored normal expression levels of these receptors.

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 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.