• Alloantigen-Induced Regulatory T Cells Generated in Presence of Vitamin C Display Enhanced Stability of Foxp3 Expression and Promote Skin Allograft Acceptance.

      Nikolouli, Eirini; Hardtke-Wolenski, Matthias; Hapke, Martin; Beckstette, Michael; Geffers, Robert; Floess, Stefan; Jaeckel, Elmar; Huehn, Jochen; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Regulatory T cells (Tregs) are critical for the maintenance of immune homeostasis and self-tolerance and can be therapeutically used for prevention of unwanted immune responses such as allotransplant rejection. Tregs are characterized by expression of the transcription factor Foxp3, and recent work suggests that epigenetic imprinting of Foxp3 and other Treg-specific epigenetic signatures genes is crucial for the stabilization of both Foxp3 expression and immunosuppressive properties within Tregs. Lately, vitamin C was reported to enhance the activity of enzymes of the ten-eleven translocation family, thereby fostering the demethylation of Foxp3 and other Treg-specific epigenetic signatures genes in developing Tregs. Here, we in vitro generated alloantigen-induced Foxp3(+) Tregs (allo-iTregs) in presence of vitamin C. Although vitamin C hardly influenced the transcriptome of allo-iTregs as revealed by RNA-seq, those vitamin C-treated allo-iTregs showed a more pronounced demethylation of Foxp3 and other Treg-specific epigenetic signatures genes accompanied with an enhanced stability of Foxp3 expression. Accordingly, when being tested in vivo in an allogeneic skin transplantation model, vitamin C-treated allo-iTregs showed a superior suppressive capacity. Together, our results pave the way for the establishment of novel protocols for the in vitro generation of alloantigen-induced Foxp3(+) Tregs for therapeutic use in transplantation medicine.
    • Clinical and Biological Manifestation of RNF168 Deficiency in Two Polish Siblings.

      Pietrucha, Barbara; Heropolitańska-Pliszka, Edyta; Geffers, Robert; Enßen, Julia; Wieland, Britta; Bogdanova, Natalia Valerijevna; Dörk, Thilo; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Germline mutations in the RING finger protein gene RNF168 have been identified in a combined immunodeficiency disorder called RIDDLE syndrome. Since only two patients have been described with somewhat different phenotypes, there is need to identify further patients. Here, we report on two Polish siblings with RNF168 deficiency due to homozygosity for a novel frameshift mutation, c.295delG, that was identified through exome sequencing. Both patients presented with immunoglobulin deficiency, telangiectasia, cellular radiosensitivity, and increased alpha-fetoprotein (AFP) levels. The younger sibling had a more pronounced neurological and morphological phenotype, and she also carried an ATM gene mutation in the heterozygous state. Immunoblot analyses showed absence of RNF168 protein, whereas ATM levels and function were proficient in lymphoblastoid cells from both patients. Consistent with the absence of RNF168 protein, 53BP1 recruitment to DNA double-strand breaks (DSBs) after irradiation was undetectable in lymphoblasts or primary fibroblasts from either of the two patients. γH2AX foci accumulated normally but they disappeared with significant delay, indicating a severe defect in DSB repair. A comparison with the two previously identified patients indicates immunoglobulin deficiency, cellular radiosensitivity, and increased AFP levels as hallmarks of RNF168 deficiency. The variability in its clinical expression despite similar cellular phenotypes suggests that some manifestations of RNF168 deficiency may be modified by additional genetic or epidemiological factors.
    • Type I Interferon Signaling Is Required for CpG-Oligodesoxynucleotide-Induced Control of Leishmania major, but Not for Spontaneous Cure of Subcutaneous Primary or Secondary L. major Infection.

      Schleicher, Ulrike; Liese, Jan; Justies, Nicole; Mischke, Thomas; Haeberlein, Simone; Sebald, Heidi; Kalinke, Ulrich; Weiss, Siegfried; Bogdan, Christian; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018)
      We previously showed that in mice infected with Leishmania major type I interferons (IFNs) initiate the innate immune response to the parasite at day 1 and 2 of infection. Here, we investigated which type I IFN subtypes are expressed during the first 8 weeks of L. major infection and whether type I IFNs are essential for a protective immune response and clinical cure of the disease. In self-healing C57BL/6 mice infected with a high dose of L. major, IFN-α4, IFN-α5, IFN-α11, IFN-α13, and IFN-β mRNA were most prominently regulated during the course of infection. In C57BL/6 mice deficient for IFN-β or the IFN-α/β-receptor chain 1 (IFNAR1), development of skin lesions and parasite loads in skin, draining lymph node, and spleen was indistinguishable from wild-type (WT) mice. In line with the clinical findings, C57BL/6 IFN-β-/-, IFNAR1-/-, and WT mice exhibited similar mRNA expression levels of IFN-γ, interleukin (IL)-4, IL-12, IL-13, inducible nitric oxide synthase, and arginase 1 during the acute and late phase of the infection. Also, myeloid dendritic cells from WT and IFNAR1-/- mice produced comparable amounts of IL-12p40/p70 protein upon exposure to L. major in vitro. In non-healing BALB/c WT mice, the mRNAs of IFN-α subtypes (α2, α4, α5, α6, and α9) were rapidly induced after high-dose L. major infection. However, genetic deletion of IFNAR1 or IFN-β did not alter the progressive course of infection seen in WT BALB/c mice. Finally, we tested whether type I IFNs and/or IL-12 are required for the prophylactic effect of CpG-oligodesoxynucleotides (ODN) in BALB/c mice. Local and systemic administration of CpG-ODN 1668 protected WT and IFN-β-/- mice equally well from progressive leishmaniasis. By contrast, the protective effect of CpG-ODN 1668 was lost in BALB/c IFNAR1-/- (despite a sustained suppression of IL-4) and in BALB/c IL-12p35-/- mice. From these data, we conclude that IFN-β and IFNAR1 signaling are dispensable for a curative immune response to L. major in C57BL/6 mice and irrelevant for disease development in BALB/c mice, whereas IL-12 and IFN-α subtypes are essential for the disease prevention by CpG-ODNs in this mouse strain.