Georg Stary

Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells

Imiquimod (IMQ), a synthetic agonist to Toll-like receptor (TLR) 7, is being successfully used for the treatment of certain skin neoplasms, but the exact mechanisms by which this compound induces tumor regression are not yet understood. While treating basal cell carcinoma (BCC) patients with topical IMQ, we detected, by immunohistochemistry, sizable numbers of both myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) within the inflammatory infiltrate. Surprisingly, peritumoral mDCs stained positive for perforin and granzyme B, whereas infiltrating pDCs expressed tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). The biological relevance of this observation can be deduced from our further findings that peripheral blood–derived CD11c+ mDCs acquired antiperforin and anti–granzyme B reactivity upon TLR7/8 stimulation and could use these molecules to effectively lyse major histocompatibility complex (MHC) class Ilo cancer cell lines. The same activation protocol led pDCs to kill MHC class I–bearing Jurkat cells in a TRAIL-dependent fashion. While suggesting that mDCs and pDCs are directly involved in the IMQ-induced destruction of BCC lesions, our data also add a new facet to the functional spectrum of DCs, ascribing to them a major role not only in the initiation but also in the effector phase of the immune response.

The PKC inhibitor AEB071 may be a therapeutic option for psoriasis

PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.

A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells

The right combination for protection Despite its prevalence, no vaccine exists to protect against infection with the sexually transmitted bacterium Chlamydia trachomatis. Stary et al. now report on one potential vaccine candidate (see the Perspective by Brunham). Vaccinating with an ultraviolet light-inactivated C. trachomatis linked to adjuvant-containing charged nanoparticles protected female conventional and humanized mice against C. trachomatis infection. The vaccine conferred protection only when delivered through mucosal routes. Protection relied on targeting the bacteria to a particular population of immunogenic dendritic cells and inducing memory T cells that resided in the female genital tract. Science, this issue 10.1126/science.aaa8205; see also p. 1322 A nanoparticle-based vaccine protects mice against infection with Chlamydia trachomatis. [Also see Perspective by Brunham] INTRODUCTION Administering vaccines through nonmucosal routes often leads to poor protection against mucosal pathogens, presumably because such vaccines do not generate memory lymphocytes that migrate to mucosal surfaces. Although mucosal vaccination induces mucosa-tropic memory lymphocytes, few mucosal vaccines are used clinically; live vaccine vectors pose safety risks, whereas killed pathogens or molecular antigens are usually weak immunogens when applied to intact mucosa. Adjuvants can boost immunogenicity; however, most conventional mucosal adjuvants have unfavorable safety profiles. Moreover, the immune mechanisms of protection against many mucosal infections are poorly understood. RATIONALE One case in point is Chlamydia trachomatis (Ct), a sexually transmitted intracellular bacterium that infects >100 million people annually. Mucosal Ct infections can cause female infertility and ectopic pregnancies. Ct is also the leading cause of preventable blindness in developing countries and induces pneumonia in infants. No approved vaccines exist to date. Here, we describe a Ct vaccine composed of ultraviolet light–inactivated Ct (UV-Ct) conjugated to charge-switching synthetic adjuvant nanoparticles (cSAPs). After immunizing mice with live Ct, UV-Ct, or UV-Ct–cSAP conjugates, we characterized mucosal immune responses to uterine Ct rechallenge and dissected the underlying cellular mechanisms. RESULTS In previously uninfected mice, Ct infection induced protective immunity that depended on CD4 T cells producing the cytokine interferon-γ, whereas uterine exposure to UV-Ct generated tolerogenic Ct-specific regulatory T cells, resulting in exacerbated bacterial burden upon Ct rechallenge. In contrast, mucosal immunization with UV-Ct–cSAP elicited long-lived protection. This differential effect of UV-Ct–cSAP versus UV-Ct was because the former was presented by immunogenic CD11b+CD103– dendritic cells (DCs), whereas the latter was presented by tolerogenic CD11b–CD103+ DCs. Intrauterine or intranasal vaccination, but not subcutaneous vaccination, induced genital protection in both conventional and humanized mice. Regardless of vaccination route, UV-Ct–cSAP always evoked a robust systemic memory T cell response. However, only mucosal vaccination induced a wave of effector T cells that seeded the uterine mucosa during the first week after vaccination and established resident memory T cells (TRM cells). Without TRM cells, mice were suboptimally protected, even when circulating memory cells were abundant. Optimal Ct clearance required both early uterine seeding by TRM cells and infection-induced recruitment of a second wave of circulating memory cells. CONCLUSIONS Mucosal exposure to both live Ct and inactivated UV-Ct induces antigen-specific CD4 T cell responses. While immunogenic DCs present the former to promote immunity, the latter is instead targeted to tolerogenic DCs that exacerbate host susceptibility to Ct infection. By combining UV-Ct with cSAP nanocarriers, we have redirected noninfectious UV-Ct to immunogenic DCs and achieved long-lived protection. This protective vaccine effect depended on the synergistic action of two memory T cell subsets with distinct differentiation kinetics and migratory properties. The cSAP technology offers a platform for efficient mucosal immunization that may also be applicable to other mucosal pathogens. Protection against C. trachomatis infection after mucosal UV-Ct–cSAP vaccination. Upon mucosal vaccination, dendritic cells carry UV-Ct–cSAP to lymph nodes and stimulate CD4 T cells. Effector T cells are imprinted to traffic to uterine mucosa (first wave) and establish tissue-resident memory cells (TRM cells). Vaccination also generates circulating memory T cells. Upon genital Ct infection, local reactivation of uterine TRM cells triggers the recruitment of the circulating memory subset (second wave). Optimal pathogen clearance requires both waves of memory cells. Genital Chlamydia trachomatis (Ct) infection induces protective immunity that depends on interferon-γ–producing CD4 T cells. By contrast, we report that mucosal exposure to ultraviolet light (UV)–inactivated Ct (UV-Ct) generated regulatory T cells that exacerbated subsequent Ct infection. We show that mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAPs) elicited long-lived protection in conventional and humanized mice. UV-Ct–cSAP targeted immunogenic uterine CD11b+CD103– dendritic cells (DCs), whereas UV-Ct accumulated in tolerogenic CD11b–CD103+ DCs. Regardless of vaccination route, UV-Ct–cSAP induced systemic memory T cells, but only mucosal vaccination induced effector T cells that rapidly seeded uterine mucosa with resident memory T cells (TRM cells). Optimal Ct clearance required both TRM seeding and subsequent infection-induced recruitment of circulating memory T cells. Thus, UV-Ct–cSAP vaccination generated two synergistic memory T cell subsets with distinct migratory properties.

Topical Treatment of Basal Cell Carcinomas in Nevoid Basal Cell Carcinoma Syndrome with a Smoothened Inhibitor

Basal cell carcinoma (BCC) is a distinctive manifestation in nevoid basal cell carcinoma syndrome (NBCCS) patients. Both inherited and acquired mutations of patched 1 (PTCH1), a tumor-suppressor gene controlling the activity of Smoothened (SMO), are the primary cause of the constitutive activation of the Hedgehog (HH) pathway, leading to the emergence of BCCs in NBCCS. LDE225, a distinct, selective antagonist of SMO, showed potent inhibition of basaloid tumor nest formation and mediated regression of preformed basaloid tumors in organ cultures of skin derived from Ptch1 heterozygous knockout mice. In a double-blind, randomized, vehicle-controlled, intraindividual study, a total of 8 NBCCS patients presenting 27 BCCs were treated twice daily with 0.75% LDE225 cream or vehicle for 4 weeks. Application of 0.75% LDE225 cream was well tolerated and showed no skin irritation. Of 13 LDE225-treated BCCs, 3 showed a complete, 9 a partial, and only 1 no clinical response. Except for one partial response, the vehicle produced no clinical response in any of the 14 treated BCCs. Treatment with 0.75% LDE225 cream in NBCCS patients was very well tolerated and caused BCC regression, thus potentially offering an attractive therapeutic alternative to currently available therapies for this indication.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.

Plasmacytoid dendritic cells express TRAIL and induce CD4 T-cell apoptosis in HIV-1 viremic patients

Artificial Toll-like receptor 7/8 (TLR7/8) ligands can endow plasmacytoid dendritic cells (pDCs) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent lytic properties. Keeping in mind that ssRNA serves as natural TLR7/8 ligand, we searched for TRAIL-expressing cells in persons infected with HIV and identified TRAIL+ pDCs in HIV-1 viremic persons, but not in nonviremic and healthy persons. TRAIL expression on pDCs was directly correlated with individual viral loads. Conversely, HIV-1 viremia was found to be associated with the up-regulation of the apoptosis-transmitting receptor TRAIL R1 on activated CD4+ T cells. As a consequence, the latter became susceptible to TRAIL-dependent pDC-mediated killing. In contrast, initiation of antiretroviral therapy led to the up-regulation of apoptosis-inhibiting TRAIL R4 on CD4+ T cells, which subsequently became resistant against pDC-mediated cellular injury. Definition of pDCs as killers of CD4+ T cells implies a new mechanism of disease progression in HIV infection.

CD4+ T Cells Are Necessary and Sufficient To Confer Protection against Chlamydia trachomatis Infection in the Murine Upper Genital Tract

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Chlamydia infections that ascend to the upper genital tract can persist, trigger inflammation, and result in serious sequelae such as infertility. However, mouse models in which the vaginal vault is inoculated with C. trachomatis do not recapitulate the course of human disease. These intravaginal infections of the mouse do not ascend efficiently to the upper genital tract, do not cause persistent infection, do not induce significant inflammation, and do not induce significant CD4+ T cell infiltration. In this article, we describe a noninvasive transcervical infection model in which we bypass the cervix and directly inoculate C. trachomatis into the uterus. We show that direct C. trachomatis infection of the murine upper genital tract stimulates a robust Chlamydia-specific CD4+ T cell response that is both necessary and sufficient to clear infection and provide protection against reinfection.

Diverse T-cell responses characterize the different manifestations of cutaneous graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HCT) and can present in an acute (aGVHD), a chronic lichenoid (clGVHD), and a chronic sclerotic form (csGVHD). It is unclear whether similar or different pathomechanisms lead to these distinct clinical presentations. To address this issue, we collected lesional skin biopsies from aGVHD (n = 25), clGVHD (n = 17), and csGVHD (n = 7) patients as well as serial nonlesional biopsies from HCT recipients (prior to or post-HCT) (n = 14) and subjected them to phenotypic and functional analyses. Our results revealed striking differences between aGVHD and clGVHD. In aGVHD, we found a clear predominance of T helper (Th)2 cytokines/chemokines and, surprisingly, of interleukin (IL)-22 messenger RNA as well as an increase of IL-22-producing CD4(+) T cells. Thymic stromal lymphopoietin, a cytokine skewing the immune response toward a Th2 direction, was elevated at day 20 to 30 post-HCT in the skin of patients who later developed aGVHD. In sharp contrast to aGVHD, the immune response occurring in clGVHD showed a mixed Th1/Th17 signature with upregulated Th1/Th17 cytokine/chemokine transcripts and elevated numbers of interferon-γ- and IL-17-producing CD8(+) T cells. Our findings shed new light on the T-cell responses involved in the different manifestations of cutaneous GVHD and identify molecular signatures indicating the development of the disease.

Glucocorticosteroids Modify Langerhans Cells To Produce TGF-β and Expand Regulatory T Cells

Although glucocorticosteroids (GCSs) have been used for many decades in transplantation and (auto)inflammatory diseases, the exact mechanisms responsible for their immunosuppressive properties are not fully understood. The purpose of this study was to characterize the effects of oral GCSs on the cutaneous immune response. We analyzed, by immunofluorescence staining and quantitative RT-PCR, residual skin biopsy material from a clinical study in which we had used oral GCS as positive control for determining the effects of candidate anti-inflammatory compounds on epicutaneous patch tests of Ni-allergic patients. Expectedly, oral GCS treatment led to a reduction of clinical symptoms and infiltrating leukocytes. Notably, we observed increased numbers of dermal FOXP3+CD25+ T cells and epidermal Langerhans cells (LCs) that were associated with upregulated mRNA expression of TGF-β in lesions of GCS-treated Ni-allergic patients. To investigate this phenomenon further, we exposed purified LCs to GCS. They exhibited, in contrast to GCS-nonexposed LCs, 1) a more immature phenotype, 2) higher intracellular amounts of TGF-β, and 3) increased receptor activator for NF-κB expression, conditions that reportedly favor the expansion of regulatory T cells (Tregs). Indeed, we observed an enhancement of functionally suppressive FOXP3+ T cells when CD3+ cells were incubated with GCS-pretreated LCs. The expansion of Tregs was inhibited by TGF-β blockage alone, and their suppressive activity was neutralized by a combination of anti–TGF-β and anti–IL-10 Abs. Our data show that systemically applied GCSs endow LCs with Treg-promoting properties and thus shed new light on the mechanisms of GCS-mediated immunosuppression.

TRAIL+ Human Plasmacytoid Dendritic Cells Kill Tumor Cells In Vitro: Mechanisms of Imiquimod- and IFN-α–Mediated Antitumor Reactivity

Dendritic cells (DCs) not only exhibit the unique capacity to evoke primary immune responses, but may also acquire TLR-triggered cytotoxic activity. We and others have previously shown that TLR7/8- and TLR9-stimulated plasmacytoid DCs (pDCs) isolated from human peripheral blood express the effector molecule TRAIL. The exact mechanisms through which pDCs acquire and elicit their cytotoxic activity are still not clear. We now show that in the absence of costimulators, TRAIL induction on pDCs occurs with agonists to intracellular TLRs only and is accompanied by a phenotypic as well as functional maturation, as evidenced by a comparatively superior MLR stimulatory capacity. pDCs acquired TRAIL in an IFN-α/β–dependent fashion and, notably, TRAIL expression on pDCs could be induced by IFN-α stimulation alone. At a functional level, both TLR7/8- (imiquimod [IMQ]) and TLR9-stimulated (CpG2216) pDCs lysed Jurkat T cells in a TRAIL- and cell contact-dependent fashion. More importantly, IFN-α–activated pDCs acquired similar cytotoxic properties, independent of TLR stimulation and maturation. Both IMQ- and IFN-α–activated pDCs could also lyse certain melanoma cell lines in a TRAIL-dependent fashion. Interestingly, suboptimal doses of IMQ and IFN-α exhibited synergistic action, leading to optimal TRAIL expression and melanoma cell lysis by pDCs. Our data imply that tumor immunity in patients receiving adjuvant IMQ and/or IFN-α may involve the active participation of cytotoxic pDCs.

Dendritic Cells in Atopic Dermatitis: Expression of FcεRI on Two Distinct Inflammation-Associated Subsets

Background: Dendritic cells (DCs) represent a major portion within the infiltrate of atopic dermatitis (AD) lesions. As antigen-presenting cells they have the ability to regulate both the quantity and quality of T-cell responses and, thus, are likely to play a key role in the pathogenesis of T-cell-dominated skin diseases such as AD. Thus we sought to identify the DC repertoire occurring in AD patients. Methods: For this purpose, we phenotypically analyzed various defined DC subsets of AD patients and healthy controls in skin biopsies and peripheral blood by immunofluorescence staining. Results: In AD lesions, two inflammation-associated DC subsets with varying expression of costimulatory molecules occurred besides epidermal Langerhans cells (LCs) and dermal myeloid DCs (dmDCs) indigenously residing in normal skin: (1) CD1a+/CD1c+/FcεRI+/IgE+/CD207– myeloid DCs (mDCs) in the epidermis and dermis and (2) CD123+/BDCA-2+/CD45RA+/CD68+ plasmacytoid DCs (pDCs) in the dermis. In the peripheral blood of the patients, these cells exhibited an immature phenotype. Interestingly, we found FcεRI and cell-bound IgE to be expressed not only on myeloid, but also on plasmacytoid DCs from both the skin and peripheral blood of AD patients. Conclusions: It is tempting to speculate that the disease-regulating role of inflammatory DCs in AD is influenced by both FcεRI occupancy and their degree of maturity.