Nathalie Vanderheyde

AS04, an Aluminum Salt- and TLR4 Agonist-Based Adjuvant System, Induces a Transient Localized Innate Immune Response Leading to Enhanced Adaptive Immunity

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4′-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-κB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4+ T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

Inhibition of human dendritic cell functions by methylprednisolone.

BACKGROUND The aim of this study was to better define how glucocorticoids influence primary human T cell responses. Dendritic cells (DC*) are the most effective antigen presenting cells able to activate naive T cells. Previous studies have shown that dexamethasone impaired the function of murine DC. Here, we analyzed how methylprednisolone (MP) might affect the function and maturation of human DC. METHODS Human DC were generated from peripheral blood mononuclear cells cultured in granulocyte macrophage-colony stimulating factor and interleukin (IL)4. DC maturation was induced either by lipopolysaccharide (LPS) or by fibroblast transfected with the CD40-ligand gene (3T6-CD40L). DC phenotype was characterized by flow cytometric analysis, their cytokine production by ELISA. The ability of DC to activate naive T cells was evaluated in mixed leukocyte reactivity. RESULTS Although MP did not affect viability of DC, it enhanced their antigen uptake and down-regulated their basal expression of CD86. The expression of CD80 and CD54 by DC was slightly decreased and HLA-DR expression was not modified. MP prevented LPS-induced DC maturation as assessed by the inhibition of CD86, CD80 and CD54 up-regulation, CD83 induction and production of TNF-alpha, IL-6, and IL-12. In contrast, when DC were stimulated by 3T6-CD40L, MP prevented only the synthesis of IL-12. Moreover, MP-treated DC were deficient in their ability to elicit proliferative responses of CD4+CD45RA+ allogeneic T cells as well as their synthesis of interferon (IFN)-gamma, IL-5, and IL-13. CONCLUSION. Glucocorticoids exert potent suppressive effects on human DC and thereby inhibit the induction of primary T cell responses.

Tumoricidal activity of monocyte-derived dendritic cells: Evidence for a caspase-8-dependent, Fas-associated death domain-independent mechanism.

Monocyte-derived dendritic cells (DC) were found to be cytotoxic for several tumor cell lines including Jurkat cells, which were killed through a calcium-independent pathway. K562 cells were resistant, excluding a NK cell-like activity. DC-mediated apoptosis did not involve classical death receptors because it was not reversed by blocking TNF/TNFR, CD95/CD95 ligand, or TNF-related apoptosis-inducing ligand/TNF-related apoptosis-inducing ligand receptor interactions. Fas-associated death domain-deficient, but not caspase-8 deficient, Jurkat cells were killed by DC. Indeed, caspase-8 cleavage was demonstrated in Jurkat cells cocultured with DC, and the use of specific caspase inhibitors confirmed that apoptosis triggered by DC was caspase-8 dependent. Furthermore, the involvement of Bcl-2 family members in the control of DC-mediated apoptosis was demonstrated by Bid cleavage in Jurkat cells cocultured with DC and resistance of Jurkat cells overexpressing Bcl-2 to DC-mediated cytotoxicity. Overall, these data indicate that monocyte-derived DC exert a caspase-8-dependent, Fas associated death domain-independent tumoricidal activity, a finding that could be relevant to their therapeutic use in cancer.

Antigen-Specific Central Memory CD4 + T Lymphocytes Produce Multiple Cytokines and Proliferate In Vivo in Humans

The function of Ag-specific central (TCM) and effector (TEM) memory CD4+ T lymphocytes remains poorly characterized in vivo in humans. Using CD154 as a marker of Ag-specific CD4+ T cells, we studied the differentiation of memory subsets following anti-hepatitis B immunization. Hepatitis B surface Ag (HBs)-specific memory CD4+ T cells were heterogeneous and included TCM (CCR7+CD27+) and TEM (CCR7−CD27+/−). HBs-specific TCM and TEM shared the capacity to produce multiple cytokines, including IL-2 and IFN-γ. Several years postimmunization, ∼10% of HBs-specific memory CD4+ T cells were in cycle (Ki67+) and the proliferating cells were CCR7+. These results suggest that the model of functional specialization of TCM and TEM cannot be applied to protein vaccine Ags and support the concept that TCM are capable of self-renewal and contribute to maintain the pool of memory cells.

Characterization of a subset of antigen-specific human central memory CD4+ T lymphocytes producing effector cytokines.

CCR7+ central memory (TCM) CD4+ T cells play a central role in long‐term immunological memory. Recent reports indicate that a proportion of CD4+ TCM is able to produce effector cytokines. The phenotype and the role of this subset remain unknown. We characterized cytokine‐producing human CD4+ TCM specific for cleared protein and persistent viral Ag. Our results demonstrate that the type of Ag stimulation is a major determinant of CD4+ TCM differentiation. CMV‐specific TCM were significantly more differentiated than protein Ag‐specific TCM and included higher proportions of IFN‐γ‐producing cells. The expression of killer cell lectin‐like receptor G1 (KLRG1) by protein Ag‐ and CMV‐specific TCM was associated with increased production of effector cytokines. KLRG1+ TCM expressed high levels of CD127, suggesting that they can survive long term under the influence of IL‐7. The induction of KLRG1+ TCM may therefore represent an important target of vaccination against pathogens controlled by cellular immune responses.