Expression of the Serpin Serine Protease Inhibitor 6 Protects Dendritic Cells from Cytotoxic T Lymphocyte–Induced Apoptosis: Differential Modulation by T Helper Type 1 and Type 2 Cells

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis

Maturation of monocyte-derived dendritic cells with Toll-like receptor 3 and 7/8 ligands combined with prostaglandin E2 results in high interleukin-12 production and cell migration

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting profile, DC are able to skew the immune response in a specific direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the effects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E2 (PGE2) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE2 to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter

Neutralization of IL-8 prevents the induction of dermatologic adverse events associated with the inhibition of epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) inhibitors are widely used in the treatment of cancer. EGFR-targeted treatment is known to be associated with a high incidence of dermatological adverse reactions, including papulopustular rash, which can be dose-limiting and may affect compliance to treatment. Currently, the pathways involved in EGFR inhibitor-induced rash are poorly understood and few treatment options for this adverse event are available. Here, we developed a model for induction of papulopustular rash in healthy human volunteers by subcutaneous injection of the anti-EGFR monoclonal antibody zalutumumab. The injection sites and surrounding skin were evaluated by a dermatologist for the presence or absence of papulopustular rash and skin biopsies were taken to confirm the macroscopical findings by immunohistochemistry. Locally injected zalutumumab induced a papulopustular rash, characterized by acute follicular neutrophil-rich hair follicle inflammation, and thus mimicked adverse events induced by systemic administration of EGFR inhibitors. In this model, we tested the hypothesis that neutrophils, attracted by IL-8, play a central role in the observed rash. Indeed, concomitant local repeat dose treatment with HuMab-10F8, a neutralizing human antibody against IL-8, reduced the rash. Inhibition of IL-8 can therefore ameliorate dermatological adverse events induced by treatment with EGFR inhibitors

Expression of the murine CD27 ligand CD70 in vitro and in vivo

The interaction between TNFR family member CD27 and its ligand CD70 promotes lymphocyte expansion and effector cell formation. In humans, control of CD27 function is partly regulated by the restricted expression of CD70. We used newly developed mAbs to characterize murine (m) CD70 expression in vitro and in vivo. On resting lymphocytes and immature dendritic cells (DC), mCD70 is absent. In vitro, Ag receptor triggering induced mCD70 mRNA in T cells, but cell surface protein expression was very low. Activated B cells synthesized much higher levels of mCD70 mRNA than activated T cells and clearly expressed mCD70 at the cell surface. mCD70 cell surface expression could also be induced on the DC line D1 and on in vitro-generated murine DC upon maturation. In lymphoid organs of naive mice, virtually no mCD70-expressing cells were found, with exception of cells in the thymic medulla, which may be epithelial in origin. However, after intranasal infection with influenza virus, lung-infiltrating T cells and T and B cells in draining lymph nodes expressed mCD70 according to immunohistology. In such activated lymphocytes, mCD70 protein is largely retained intracellularly. Plasma membrane expression of mCD70 was only detectable by flow cytometry on a small proportion of lung-infiltrating T cells and peaked at the height of the primary response. Thus, expression of CD70 in the mouse is highly regulated at the transcriptional and posttranslational level. This most likely serves to limit excessive effector cell formation after antigenic stimulatio

Expression of the murine CD27 ligand CD70 in vitro and in vivo

The interaction between TNFR family member CD27 and its ligand CD70 promotes lymphocyte expansion and effector cell formation. In humans, control of CD27 function is partly regulated by the restricted expression of CD70. We used newly developed mAbs to characterize murine (m) CD70 expression in vitro and in vivo. On resting lymphocytes and immature dendritic cells (DC), mCD70 is absent. In vitro, Ag receptor triggering induced mCD70 mRNA in T cells, but cell surface protein expression was very low. Activated B cells synthesized much higher levels of mCD70 mRNA than activated T cells and clearly expressed mCD70 at the cell surface. mCD70 cell surface expression could also be induced on the DC line D1 and on in vitro-generated murine DC upon maturation. In lymphoid organs of naive mice, virtually no mCD70-expressing cells were found, with exception of cells in the thymic medulla, which may be epithelial in origin. However, after intranasal infection with influenza virus, lung-infiltrating T cells and T and B cells in draining lymph nodes expressed mCD70 according to immunohistology. In such activated lymphocytes, mCD70 protein is largely retained intracellularly. Plasma membrane expression of mCD70 was only detectable by flow cytometry on a small proportion of lung-infiltrating T cells and peaked at the height of the primary response. Thus, expression of CD70 in the mouse is highly regulated at the transcriptional and posttranslational level. This most likely serves to limit excessive effector cell formation after antigenic stimulation

Histology and immunohistochemistry of biopsies from zalutumumab-injected skin treated with HuMab-10F8 or untreated.

<p>Data shown are representative sections of skin biopsies of one subject for part III of the study. Biopsies were taken at day 14 after two injections with 100 µg of zalutumumab with [A, C, E, G, I, K] or without HuMab-10F8 [B, D, F, H, J, L]. A, B: HE staining; C, D: staining for EGFR; E–H: staining for macrophages and I–L: staining for neutrophils. Extensive influx of inflammatory cells in the vicinity of the hair follicle with histological signs of folliculitis was observed in zalutumumab-injected skin (right panels), whereas in the zalutumumab-injected skin treated with HuMab-10F8 (left panels) inflammatory cells were virtually absent and sebaceous glands and hair follicles showed normal histological features. Inflammatory infiltrate in the zalutumumab-injected skin was mainly composed of neutrophils (neutrophil elastase positive cells, J, L) and to a lesser extent of macrophages; (CD68 positive cells F, H). Scale bars are 100 µm (A–F, I, J) and 20 µm (G, H, K, L). G, H, K and L are magnifications of the squared areas indicated in E, F, I and J.</p

Dose escalation schedule for zalutumumab injections.

<p>Every injection of zalutumumab was accompanied by a control injection of an identical volume of isotonic saline at a different site. In the third part the dose escalation started at 100 μg.</p

Histology of biopsies from saline- or zalutumumab-injected skin (part I of the study).

<p>Representative sections are shown of skin biopsies of one subject for part I of the study (following two injections of 10 μg of zalutumumab). Sections were stained with HE to show nucleated cells and (patho-)histological features. In the saline-injected skin (A, C), inflammatory cells were virtually absent and the hair follicle structure was intact. Note that after zalutumumab injection (B, D), the hair follicle structure was destroyed and extensive influx of inflammatory cells (including neutrophils) in the vicinity of the destroyed hair follicle/glandular structures can be observed (asterisks). Scale bars are 100 µm (A, B) and 20 µm (C, D).</p

Trial design.

<p>Injection schemes for the three different parts of the study are shown. Subjects were injected with different doses of zalutumumab at different sites, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039706#pone-0039706-t001" target="_blank">Table 1</a>. Sc – screening of patient.</p