Norman Nausch

NKG2D ligands in tumor immunity

The activating receptor NKG2D (natural-killer group 2, member D) and its ligands play an important role in the NK, γδ+ and CD8+ T-cell-mediated immune response to tumors. Ligands for NKG2D are rarely detectable on the surface of healthy cells and tissues, but are frequently expressed by tumor cell lines and in tumor tissues. It is evident that the expression levels of these ligands on target cells have to be tightly regulated to allow immune cell activation against tumors, but at the same time avoid destruction of healthy tissues. Importantly, it was recently discovered that another safeguard mechanism controlling activation via the receptor NKG2D exists. It was shown that NKG2D signaling is coupled to the IL-15 receptor pathway in a cell-specific manner suggesting that priming of NKG2D-mediated activation depends on the cellular microenvironment and the distinct cellular context. This review will provide a broad overview of our up-to-date knowledge of the NKG2D receptor and its ligands in the context of tumor immunology. Strategies to amplify NKG2D-mediated antitumor responses and counteract tumor immune escape mechanisms will be discussed.

Mononuclear myeloid-derived suppressor cells express RAE-1 and activate natural killer cells

Myeloid-derived suppressor cells (MDSCs) accumulate in cancer patients and tumor-bearing mice and potently suppress T-cell activation. In this study, we investigated whether MDSCs regu-late natural killer (NK)-cell function. We discovered that mononuclear Gr-1(+)CD11b(+)F4/80(+) MDSCs isolated from RMA-S tumor-bearing mice do not suppress, but activate NK cells to produce high amounts of IFN-gamma. Gr-1(+)CD11b(+)F4/80(+) MDSCs isolated from tumor-bearing mice, but not myeloid cells from naive mice, expressed the ligand for the activating receptor NKG2D, RAE-1. NK-cell activation by MDSCs depended partially on the interaction of NKG2D on NK cells with RAE-1 on MDSCs. NK cells eliminated Gr-1(+)CD11b(+)F4/80(+) MDSCs in vitro and upon adoptive transfer in vivo. Finally, depletion of Gr-1(+) cells that comprise MDSCs confirmed their protective role against the NK-sensitive RMA-S lymphoma in vivo. Our study reveals that MDSCs do not suppress all aspects of antitumor immune responses and defines a novel, unexpected activating role of MDSCs on NK cells. Thus, our results have great impact on the design of immune therapies against cancer aiming at the manipulation of MDSCs.

Interferon-γ down-regulates NKG2D ligand expression and impairs the NKG2D-mediated cytolysis of MHC class I-deficient melanoma by natural killer cells

NKG2D operates as an activating receptor on natural killer (NK) cells and costimulates the effector function of αβ CD8+ T cells. Ligands of NKG2D, the MHC class I chain‐related (MIC) and UL16 binding protein (ULBP) molecules, are expressed on a variety of human tumors, including melanoma. Recent studies in mice demonstrated that NKG2D mediates tumor immune surveillance, suggesting that antitumor immunity in humans could be enhanced by therapeutic manipulation of NKG2D ligand (NKG2DL) expression. However, signals and mechanisms regulating NKG2DL expression still need to be elucidated. Here, we asked whether the proinflammatory cytokine Interferon‐γ (IFN‐γ) affects NKG2DL expression in melanoma. Cell lines, established from MHC class I‐negative and ‐positive melanoma metastases, predominantly expressed MICA and ULBP2 molecules on their surface. Upon IFN‐γ treatment, expression of MICA, in some cases, also of ULBP2 decreased. Besides melanoma, this observation was made also for glioma cells. Down‐regulation of NKG2DL surface expression was dependent on the cytokine dose and the duration of treatment, but was neither due to an intracellular retention of the molecules nor to an increased shedding of ligands from the tumor cell surface. Instead, quantitative RT‐PCR revealed a decrease of MICA‐specific mRNA levels upon IFN‐γ treatment and siRNA experiments pointed to an involvement of STAT‐1 in this process. Importantly, IFN‐γ‐treated MHC class I‐negative melanoma cells were less susceptible to NKG2D‐mediated NK cell cytotoxicity. Our study suggests that IFN‐γ, by down‐regulating ligand expression, might facilitate escape of MHC class I‐negative melanoma cells from NKG2D‐mediated killing by NK cells.

Parasite-Derived MicroRNAs in Host Serum As Novel Biomarkers of Helminth Infection

Background MicroRNAs (miRNAs) are a class of short non-coding RNA that play important roles in disease processes in animals and are present in a highly stable cell-free form in body fluids. Here, we examine the capacity of host and parasite miRNAs to serve as tissue or serum biomarkers of Schistosoma mansoni infection. Methods/Principal Findings We used Exiqon miRNA microarrays to profile miRNA expression in the livers of mice infected with S. mansoni at 7 weeks post-infection. Thirty-three mouse miRNAs were differentially expressed in infected compared to naïve mice (>2 fold change, p<0.05) including miR-199a-3p, miR-199a-5p, miR-214 and miR-21, which have previously been associated with liver fibrosis in other settings. Five of the mouse miRNAs were also significantly elevated in serum by twelve weeks post-infection. Sequencing of small RNAs from serum confirmed the presence of these miRNAs and further revealed eleven parasite-derived miRNAs that were detectable by eight weeks post infection. Analysis of host and parasite miRNA abundance by qRT-PCR was extended to serum of patients from low and high infection sites in Zimbabwe and Uganda. The host-derived miRNAs failed to distinguish uninfected from infected individuals. However, analysis of three of the parasite-derived miRNAs (miR-277, miR-3479-3p and bantam) could detect infected individuals from low and high infection intensity sites with specificity/sensitivity values of 89%/80% and 80%/90%, respectively. Conclusions This work identifies parasite-derived miRNAs as novel markers of S. mansoni infection in both mice and humans, with the potential to be used with existing techniques to improve S. mansoni diagnosis. In contrast, although host miRNAs are differentially expressed in the liver during infection their abundance levels in serum are variable in human patients and may be useful in cases of extreme pathology but likely hold limited value for detecting prevalence of infection.

Schistosoma haematobium treatment in 1-5 year old children: safety and efficacy of the antihelminthic drug praziquantel

Background Morbidity due to schistosomiasis is currently controlled by treatment of schistosome infected people with the antihelminthic drug praziquantel (PZQ). Children aged up to 5 years are currently excluded from schistosome control programmes largely due to the lack of PZQ safety data in this age group. This study investigated the safety and efficacy of PZQ treatment in such children. Methods Zimbabwean children aged 1–5 years (n = 104) were treated with PZQ tablets and side effects were assessed by questionnaire administered to their caregivers within 24 hours of taking PZQ. Treatment efficacy was determined 6 weeks after PZQ administration through schistosome egg counts in urine. The change in infection levels in the children 1–5 years old (n = 100) was compared to that in 6–10 year old children (n = 435). Principal Findings Pre-treatment S. haematobium infection intensity in 1–5 year olds was 14.6 eggs/10 ml urine and prevalence was 21%. Of the 104 children, 3.8% reported side effects within 24 hours of taking PZQ treatment. These were stomach ache, loss of appetite, lethargy and inflammation of the face and body. PZQ treatment significantly reduced schistosome infection levels in 1–5 year olds with an egg reduction rate (ERR) of 99% and cure rate (CR) of 92%. This was comparable to the efficacy of praziquantel in 6–10 year olds where ERR was 96% and CR was 67%. Interpretation/Significance PZQ treatment is as safe and efficacious in children aged 1–5 years as it is in older children aged 6–10 years in whom PZQ is the drug of choice for control of schistosome infections.

Regulatory and Activated T Cells in Human Schistosoma haematobium Infections

Acquired immunity against helminths is characterised by a complex interplay between the effector Th1 and Th2 immune responses and it slowly manifests with age as a result of cumulative exposure to parasite antigens. Data from experimental models suggest that immunity is also influenced by regulatory T cells (Treg), but as yet studies on Treg in human schistosome infections are limited. This study investigated the relationship between schistosome infection intensity and the two cell populations regulatory T cells (Treg: CD4+(dim)CD25+(high)FOXP3+CD127low), and activated (Tact: CD4+CD25+FOXP3−) T cells in Zimbabweans exposed to Schistosoma haematobium parasites. Participants were partitioned into two age groups, young children (8–13 years) in whom schistosome infection levels were rising to peak and older people (14+ years) with declining infection levels. The relationship between Tact proportions and schistosome infection intensity remained unchanged with age. However Treg proportions rose significantly with increasing infection in the younger age group. In contrast Treg were negatively correlated to infection intensity in the older age group. The relative proportions of regulatory T cells differ significantly between young individuals in whom high infection is associated with an enhanced regulatory phenotype and older infected patients in whom the regulatory response is attenuated. This may influence or reflect different stages of the development of protective schistosome acquired immunity and immunopathogenesis.

Schistosome infection intensity is inversely related to auto-reactive antibody levels.

In animal experimental models, parasitic helminth infections can protect the host from auto-immune diseases. We conducted a population-scale human study investigating the relationship between helminth parasitism and auto-reactive antibodies and the subsequent effect of anti-helminthic treatment on this relationship. Levels of antinuclear antibodies (ANA) and plasma IL-10 were measured by enzyme linked immunosorbent assay in 613 Zimbabweans (aged 2–86 years) naturally exposed to the blood fluke Schistosoma haematobium. ANA levels were related to schistosome infection intensity and systemic IL-10 levels. All participants were offered treatment with the anti-helminthic drug praziquantel and 102 treated schoolchildren (5–16 years) were followed up 6 months post-antihelminthic treatment. ANA levels were inversely associated with current infection intensity but were independent of host age, sex and HIV status. Furthermore, after allowing for the confounding effects of schistosome infection intensity, ANA levels were inversely associated with systemic levels of IL-10. ANA levels increased significantly 6 months after anti-helminthic treatment. Our study shows that ANA levels are attenuated in helminth-infected humans and that anti-helminthic treatment of helminth-infected people can significantly increase ANA levels. The implications of these findings are relevant for understanding both the aetiology of immune disorders mediated by auto-reactive antibodies and in predicting the long-term consequences of large-scale schistosomiasis control programs.

Cutting Edge: The AP-1 Subunit JunB Determines NK Cell-Mediated Target Cell Killing by Regulation of the NKG2D-Ligand RAE-1ε

The activating receptor NKG2D and its ligands RAE-1 play an important role in the NK, γδ+, and CD8+ T cell-mediated immune response to tumors. Expression levels of RAE-1 on target cells have to be tightly controlled to allow immune cell activation against tumors but to avoid destruction of healthy tissues. In this study, we report that cell surface expression of RAE-1ε is greatly enhanced on cells lacking JunB, a subunit of the transcription complex AP-1. Furthermore, tissue-specific junB knockout mice respond to 12-O-tetradecanoyl-phorbol-13-acetate, a potent AP-1 activator, with markedly increased and sustained epidermal RAE-1ε expression. Accordingly, junB-deficient cells are efficiently killed via NKG2D by NK cells and induce IFN-γ production. Our data indicate that the transcription factor AP-1, which is involved in tumorigenesis and cellular stress responses, regulates RAE-1ε. Thus, up-regulated RAE-1ε expression due to low levels of JunB could alert immune cells to tumors and stressed cells.

Atopy is inversely related to schistosome infection intensity: a comparative study in Zimbabwean villages with distinct levels of Schistosoma haematobium infection.

Background: The hygiene hypothesis suggests that parasitic infections protect against allergic diseases by modulating the host’s immune responses. Experimental studies indicate that this protection depends on the intensity of parasitic infection, but this observation has not been tested in human populations. The aim of this study is to investigate whether the intensity of Schistosoma haematobium infection is related to atopic responses and whether this relationship differs between populations with distinct parasite transmission dynamics. Methods: The study was conducted in two villages with different Schistosoma haematobium transmission dynamics, i.e. high (n = 365) and low (n = 307) transmission. Allergic reactivity to the common house dust mite (Dermatophagoides pteronyssinus) was measured by skin prick tests and allergen-specific IgE and IgG4 quantified by enzyme-linked immunosorbent assay. Atopic responses were related to current infection intensity and schistosome transmission levels. Results: Schistosome infection intensity was negatively associated with the skin prick reactivity, mite-specific IgE and the ratio IgE/IgG4 in the high-transmission village. However, when only low levels of infection were analyzed in the 2 villages, there was no correlation between mite-specific responses and infection intensity. Conclusion: The relationship between schistosome infection and atopic responses is dependent on the intensity of current schistosome infection. Thus, consistent with results from animal models, with an increasing parasite burden, the immunoregulation of immune responses to allergens appears to become more pronounced.

Circulating cytokine levels and antibody responses to human Schistosoma haematobium: IL-5 and IL-10 levels depend upon age and infection status.

Experimental schistosome infections induce strong parasite‐specific Th2 responses. This study aims to relate human systemic cytokine and antibody levels to schistosome infection levels and history. Levels of anti‐Schistosoma haematobium antibodies (directed against crude cercariae, egg and adult worm antigens) and plasma cytokines (IFN‐γ, IL‐2, IL‐4, IL‐5, IL‐10, IL‐13, IL‐17, IL‐21, and IL‐23) were measured by ELISA in 227 Zimbabweans (6–60 years old) in a schistosome‐endemic area and related to age and infection status. Egg‐positive people had significantly higher levels of specific antibodies, IL‐2, IFN‐γ and IL‐23. In contrast, egg‐negative individuals had significantly higher circulating IL‐10, IL‐4, IL‐13 and IL‐21 that were detected with high frequency in all participants. Subjects with detectable plasma IL‐17 produced few or no eggs. When analyzed by age, IL‐4 and IL‐10 increased significantly, as did schistosome‐specific antibodies. However, when age was combined with infection status, IL‐5 declined over time in egg‐positive people, while increased with age in the egg‐negative group. Older, lifelong residents had significantly higher IL‐4 and IL‐5 levels than younger egg‐negative people. Thus, a mixed Th1/Th2 systemic environment occurs in people with patent schistosome infection, while a stronger Th2‐dominated suite of cytokines is evident in egg‐negative individuals.