Karin Loser

Frontline: Neuropilin-1: a surface marker of regulatory T cells

CD4+CD25+ regulatory T cells (Treg cells) control immune responsiveness to a large variety of antigens. The isolation and therapeutic manipulation of Treg cells requires the use of reliable surface receptors that are selectively up‐regulated in Treg cells. On the basis of global gene expression studies, we identified neuropilin‐1 (Nrp1) as a specific surface marker for CD4+CD25+ Treg cells. Nrp1, a receptor involved in axon guidance, angiogenesis, and the activation of T cells, is constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status. In contrast, Nrp1 expression is down‐regulated in naive CD4+CD25– T cells after TCR stimulation. Furthermore, CD4+Nrp1high T cells express high levels of Foxp3 and suppress CD4+CD25– T cells. Thus, Nrp1 constitutes a useful surface marker to distinguish Treg cells from both naive and recently activated CD4+CD25+ non‐regulatory T cells.

Epidermal RANKL controls regulatory T-cell numbers via activation of dendritic cells.

Regulatory CD4+CD25+ T cells are important in suppressing immune responses. The requirements for the maintenance of peripheral CD4+CD25+ T cells remain incompletely understood. Receptor activator of NF-κB (RANK) and its ligand (RANKL; also known as CD254, OPGL and TRANCE) are key regulators of bone remodeling, mammary gland formation, lymph node development and T-cell/dendritic cell communication. Here we report that RANKL is expressed in keratinocytes of the inflamed skin. RANKL overexpression in keratinocytes resulted in functional alterations of epidermal dendritic cells and systemic increases of regulatory CD4+CD25+ T cells. Thus, epidermal RANKL expression can change dendritic cell functions to maintain the number of peripheral CD4+CD25+ regulatory T cells. Epidermal RANKL mediated ultraviolet-induced immunosuppression and overexpression of epidermal RANKL suppressed allergic contact hypersensitivity responses and the development of systemic autoimmunity. Therefore, environmental stimuli at the skin can rewire the local and systemic immune system by means of RANKL.

The Toll-like receptor 4 ligands Mrp8 and Mrp14 are crucial in the development of autoreactive CD8+ T cells

Mechanisms linking innate immunity and autoimmune responses are poorly understood. Myeloid-related protein-8 (Mrp8) and Mrp14 are damage-associated molecular pattern molecules (DAMPs) highly upregulated in various autoimmune disorders. We show in a mouse autoimmune model that local Mrp8 and Mrp14 production is essential for the induction of autoreactive CD8+ T cells and the development of systemic autoimmunity. This effect is mediated via Toll-like receptor 4 (TLR4) signaling leading to increased interleukin-17 (IL-17) expression. Notably, expression of Mrp8 and Mrp14 was upregulated in cutaneous lupus erythematosus, and stimulation of CD8+ T cells from individuals with lupus erythematosus with MRP proteins resulted in an upregulation of IL-17, suggesting a key role for MRP8 and MRP14 for the development of autoreactive lymphocytes during human autoimmunity as well. These results demonstrate a link between local expression of DAMP molecules and the development of systemic autoimmunity.

Endothelial basement membrane laminin |[alpha]|5 selectively inhibits T lymphocyte extravasation into the brain

Specific inhibition of the entry of encephalitogenic T lymphocytes into the central nervous system in multiple sclerosis would provide a means of inhibiting disease without compromising innate immune responses. We show here that targeting lymphocyte interactions with endothelial basement membrane laminins provides such a possibility. In mouse experimental autoimmune encephalomyelitis, T lymphocyte extravasation correlates with sites expressing laminin α4 and small amounts of laminin α5. In mice lacking laminin α4, laminin α5 is ubiquitously expressed along the vascular tree, resulting in marked and selective reduction of T lymphocyte infiltration into the brain and reduced disease susceptibility and severity. Vessel phenotype and immune response were not affected in these mice. Rather, laminin α5 directly inhibited integrin α6β1–mediated migration of T lymphocytes through laminin α4. The data indicate that T lymphocytes use mechanisms distinct from other immune cells to penetrate the endothelial basement membrane barrier, permitting specific targeting of this immune cell population.

IL-10 Controls Ultraviolet-Induced Carcinogenesis in Mice

UV radiation-induced immunosuppression contributes significantly to the development of UV-induced skin cancer by inhibiting protective immune responses. IL-10 has been shown to be a key mediator of UV-induced immunosuppression. To investigate the role of IL-10 during photocarcinogenesis, groups of IL-10+/+, IL-10+/−, and IL-10−/− mice were chronically irradiated with UV. IL-10+/+ and IL-10+/− mice developed skin cancer to similar extents, whereas IL-10−/− mice were protected against the induction of skin malignancies by UV. Because UV is able to induce regulatory T cells, which play a role in the suppression of protective immunity, UV-induced regulatory T cell function was analyzed. Splenic regulatory T cells from UV-irradiated IL-10−/− mice were unable to confer immunosuppression upon transfer into naive recipients. UV-induced CD4+CD25+ T cells from IL-10−/− mice showed impaired suppressor function when cocultured with conventional CD4+CD25− T cells. CD4+CD25− T cells from IL-10−/− mice produced increased amounts of IFN-γ and enhanced numbers of CD4+TIM-3+ T cells were detectable within UV-induced tumors in IL-10−/− mice, suggesting strong Th1-drived immunity. Mice treated with CD8+ T cells from UV-irradiated IL-10−/− mice rejected a UV tumor challenge significantly faster, and augmented numbers of granzyme A+ cells were detected within injected UV tumors in IL-10−/− animals, suggesting marked antitumoral CTL responses. Together, these findings indicate that IL-10 is critically involved in antitumoral immunity during photocarcinogenesis. Moreover, these results point out the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against UV-induced tumor development.

Spontaneous tumor rejection by cbl-b–deficient CD8+ T cells

The concept of tumor surveillance implies that specific and nonspecific components of the immune system eliminate tumors in the early phase of malignancy. Understanding the biochemical mechanisms of tumor immunosurveillance is of paramount significance because it might allow one to specifically modulate spontaneous antitumor activity. We report that inactivation of the E3 ligase Casitas B cell lymphoma-b (Cbl-b) confers spontaneous in vivo rejection of tumor cells that express human papilloma virus antigens. Moreover, cbl-b−/− mice develop significantly fewer ultraviolet B (UVB)–induced skin malignancies and reject UVB-induced skin tumors. CD8+ T cells were identified as key players in the spontaneous tumor rejection response. Loss of Cbl-b not only enhances antitumor reactivity of CD8+ T cells but also occurs in the absence of CD4+ T cells. Mechanistically, cbl-b−/− CD8+ T cells are resistant to T regulatory cell–mediated suppression and exhibit enhanced activation and rapid tumor infiltration. Importantly, therapeutic transfer of naive cbl-b−/− CD8+ T cells is sufficient to mediate rejection of established tumors. Even up to 1 yr after the first encounter with the tumor cells, cbl-b−/− mice carry an “anticancer memory.” These data identify Cbl-b as a key signaling molecule that controls spontaneous antitumor activity of cytotoxic T cells in different cancer models. Inhibition of Cbl-b is a novel approach to stimulate long-lasting immunity against cancer.

Vitamin D receptor signaling contributes to susceptibility to infection with Leishmania major

We have previously reported that 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3) can selectively suppress key functions of interferon‐gamma (IFN‐γ) activated macrophages. To further explore this mechanism for its relevance in vivo, we investigated an infection model that crucially depends on the function of IFN‐γ activated macrophages, the infection with the intracellular protozoan Leishmania mαjor. 1α,25(OH)2D3 treatment of L. major infected macrophages demonstrated a vitamin D receptor (Vdr) dependent inhibition of macrophage killing activity. Further analysis showed that this was a result of decreased production of nitric oxide by 1α,25(OH)2D3‐treated macrophages due to Vdr‐dependent up‐regulation of arginase 1 ex‐pression, which overrides NO production by Nos2. When analyzing the course of infection in vivo,we found that Vdr‐knockout (Vdr‐KO) mice were more resistant to L. major infection than their wild‐type littermates. This result is in agreement with an inhibitory influence of 1α,25(OH)2D3 on the macrophage mediated host defense. Further investigation showed that Vdr‐KO mice developed an unaltered T helper cell type 1 (Th1) response on infection as indicated by normal production of IFN‐γ by CD4+ and CD8+ T cells. Therefore, we propose that the absence of 1α,25(OH)2D3‐mediated inhibition of macrophage microbicidal activity in Vdr‐KO mice results in increased resistance to Leishmania infection.

Ultraviolet B light attenuates the systemic immune response in central nervous system autoimmunity

Environmental conditions (eg, latitude) play a critical role in the susceptibility and severity of many autoimmune disorders, including multiple sclerosis (MS). Here, we investigated the mechanisms underlying the beneficial effects of immune regulatory processes induced in the skin by moderate ultraviolet B (UVB) radiation on central nervous system (CNS) autoimmunity.

Heart-directed Expression of a Human Cardiac Isoform of cAMP-Response Element Modulator in Transgenic Mice

The transcriptional activation mediated by cAMP-response element (CRE) and transcription factors of the CRE-binding protein (CREB)/CRE modulator (CREM) family represents an important mechanism of cAMP-dependent gene regulation possibly implicated in detrimental effects of chronic β-adrenergic stimulation in end-stage heart failure. We studied the cardiac role of CREM in transgenic mice with heart-directed expression of CREM-IbΔC-X, a human cardiac CREM isoform. Transgenic mice displayed atrial enlargement with atrial and ventricular hypertrophy, developed atrial fibrillation, and died prematurely. In vivo hemodynamic assessment revealed increased contractility of transgenic left ventricles probably due to a selective up-regulation of SERCA2, the cardiac Ca2+-ATPase of the sarcoplasmic reticulum. In transgenic ventricles, reduced phosphorylation of phospholamban and of the CREB was associated with increased activity of serine-threonine protein phosphatase 1. The density of β1-adrenoreceptor was increased, and messenger RNAs encoding transcription factor dHAND and small G-protein RhoB were decreased in transgenic hearts as compared with wild-type controls. Our results indicate that heart-directed expression of CREM-IbΔC-X leads to complex cardiac alterations, suggesting CREM as a central regulator of cardiac morphology, function, and gene expression.

Molecular and Cellular Mechanisms of Photocarcinogenesis

Skin cancer constitutes one of the most frequent types of malignancies in humans with rapidly increasing incidences almost worldwide. UVR is an essential risk factor for the development of premalignant as well as malignant skin lesions. In this context UVR can function as a complete carcinogen by inducing “UV signature” DNA mutations and by suppressing protective cellular antitumoral immune responses. UV‐induced DNA damage can result in impaired cutaneous cell cycle control if cell cycle regulators, such as the p53 gene, are affected. Besides interfering with cell cycle control genes, UV‐induced DNA damage can result in the release of interleukin‐10, a cytokine with known immunosuppressive effects on T‐helper(h)‐1 cells. For the development of antitumoral immune responses antigen‐specific activation of effector T cells by antigen‐presenting cells (APC) is required. It was demonstrated that UVR can inhibit antigen presentation both directly and indirectly via the induction of suppressive cytokines. In addition, subsets of T cells are induced upon UVR, which can actively suppress major histocompatibility complex class I/II‐restricted immune responses. These UV‐induced regulatory T cells appear to belong to the CD4+CD25+ T cell lineage and can express the characteristic transcription factor Foxp3, which programs for suppressor function. In mice UV‐induced regulatory T cells can control the development of UV‐induced skin cancer. Peripheral regulatory T cells are maintained by the expression of B7 molecules and can be expanded by APC of the skin. Recently, epidermal expression of CD254 (RANKL) has been shown to connect UVR with the expansion of regulatory CD4+CD25+ T cells. In the following, new molecular and cellular mechanisms of UV‐induced skin tumor development will be described and discussed.