De novo DNA methylation controls neuronal maturation during adult hippocampal neurogenesis

Adult neurogenesis enables the life-long addition of functional neurons to the hippocampus and is regulated by both cell-intrinsic molecular programs and behavioral activity. De novo DNA methylation is crucial for embryonic brain development, but its role during adult hippocampal neurogenesis has remained unknown. Here, we show that de novo DNA methylation is critical for maturation and functional integration of adult-born neurons in the mouse hippocampus. Bisulfite sequencing revealed that de novo DNA methyltransferases target neuronal enhancers and gene bodies during adult hippocampal neural stem cell differentiation, to establish neuronal methylomes and facilitate transcriptional up-regulation of neuronal genes. Inducible deletion of both de novo DNA methyltransferases Dnmt3a and Dnmt3b in adult neural stem cells did not affect proliferation or fate specification, but specifically impaired dendritic outgrowth and synaptogenesis of newborn neurons, thereby hampering their functional maturation. Consequently, abolishing de novo DNA methylation modulated activation patterns in the hippocampal circuitry and caused specific deficits in hippocampus-dependent learning and memory. Our results demonstrate that proper establishment of neuronal methylomes during adult neurogenesis is fundamental for hippocampal function

Inducible IL-7 hyperexpression influences lymphocyte homeostasis and function and increases allograft rejection.

The IL-7/IL-7R pathway is essential for lymphocyte development and disturbances in the pathway can lead to immune deficiency or T cell mediated destruction. Here, the effect of transient hyperexpression of IL-7 was investigated on immune regulation and allograft rejection under immunosuppression. An experimental in vivo immunosuppressive mouse model of IL-7 hyperexpression was developed using transgenic mice (C57BL/6 background) carrying a tetracycline inducible IL-7 expression cassette, which allowed the temporally controlled induction of IL-7 hyperexpression by Dexamethasone and Doxycycline treatment. Upon induction of IL-7, the B220+ c-kit+ Pro/Pre-B I compartment in the bone marrow increased as compared to control mice in a serum IL-7 concentration-correlated manner. IL-7 hyperexpression also preferentially increased the population size of memory CD8+ T cells in secondary lymphoid organs, and reduced the proportion of CD4+Foxp3+ T regulatory cells. Of relevance to disease, conventional CD4+ T cells from an IL-7-rich milieu escaped T regulatory cell-mediated suppression in vitro and in a model of autoimmune diabetes in vivo. These findings were validated using an IL-7/anti-IL7 complex treatment mouse model to create an IL-7 rich environment. To study the effect of IL-7 on islet graft survival in a mismatched allograft model, BALB/c mice were rendered diabetic by streptozotocin und transplanted with IL-7-inducible or control islets from C57BL/6 mice. As expected, Dexamethasone and Doxycycline treatment prolonged graft median survival as compared to the untreated control group in this transplantation mouse model. However, upon induction of local IL-7 hyperexpression in the transplanted islets, graft survival time was decreased and this was accompanied by an increased CD4+ and CD8+ T cell infiltration in the islets. Altogether, the findings show that transient elevations of IL-7 can impair immune regulation and lead to graft loss also under immune suppression

Vagaries of the ELISpot assay: Specific detection of antigen responsive cells requires purified CD8⁺ T cells and MHC class I expressing antigen presenting cell lines.

Quantification of antigen-specific CD8(+) T cells is important for monitoring infection, vaccination, and response to therapy in cancer and immune-mediated diseases. Cytokine enzyme-linked-immunospot (ELISpot) assays are often used for this purpose. We found that substantial spot formation in IFNγ ELISpot assays occurred independently of CD8(+) T cells even when classical MHC class I restricted peptides are used for stimulation. Using fractionated cells and intracellular cytokine staining, the non-CD8(+) T cell IFNγ production was attributed to the CD4(+) T cell fraction. We therefore refined a cell line-based ELISpot assay combining HLA-A*0201 expressing K562 cells for antigen presentation with purified CD8(+) T cells and demonstrated that it specifically detected CD8(+) T cell responses with detection limits comparable to traditional ELISpot assays and dextramer-based quantification. The assay was further adapted to whole antigen responses with antigen (pre-proinsulin)-expressing HLA-A*0201K562 cells. Thus, we revealed and corrected a weak spot of the CD8(+) ELISpot assay

Igrp and insulin vaccination induce CD8⁺ T cell mediated autoimmune diabetes in the RIP-CD80GP mouse.

Autoimmune diabetes is characterized by autoantigen-specific T cell-mediated destruction of pancreatic islet beta cells and CD8(+) T cells are key players during this process. We assessed whether the bitransgenic RIP-CD80 x RIP-LCMV-GP (RIP-CD80GP) mice may be a versatile antigen specific model of inducible CD8(+) T cell mediated autoimmune diabetes. Antigen-encoding DNA, peptide loaded dendritic cells, and antigen plus incomplete Freund's adjuvant were used for vaccination. Of 14 pancreatic proteins tested by DNA vaccination, murine pre-proinsulin 2 (100% of mice; median time after vaccination, 60 days), and Igrp (77%, 58 days) could induce diabetes. DNA vaccination with zinc transporter 8, Ia-2, Ia-2β, Gad67, Chromogranin A, IAPP, and Nkx2.2 induced diabetes development in 25-33% of mice, and with Gad65, Sgne1, Pdx1, Cel, glucagon, and control HBsAg in <20% of mice. Diabetes induction efficiency could be increased by DNA vaccination with a vector encoding a ubiquitin-antigen fusion construct. Diabetic mice had florid T cell islet infiltration. CD8(+) T cell targets of Igrp were identified with a peptide library based ELISpot assay, and diabetes could also be induced by vaccination with MHC class I restricted Igrp peptides loaded on mature dendritic cells. Vaccination with antigen plus incomplete Freund's adjuvant, which can prevent diabetes in other models, led to rapid diabetes development in the RIP-CD80GP mouse. We conclude that RIP-CD80GP mice are a versatile model of antigen specific autoimmune diabetes and may complement existing mouse models of autoimmune diabetes for evaluating CD8(+) T cell-targeted prevention strategies

Identification of infants with increased type 1 diabetes genetic risk for enrollment into Primary Prevention Trials-GPPAD-02 study design and first results

Primary prevention of type 1 diabetes (T1D) requires intervention in genetically at-risk infants. The Global Platform for the Prevention of Autoimmune Diabetes (GPPAD) has established a screening program, GPPAD-02, that identifies infants with a genetic high risk of T1D, enrolls these into primary prevention trials, and follows the children for beta-cell autoantibodies and diabetes. Genetic testing is offered either at delivery, together with the regular newborn testing, or at a newborn health care visits before the age of 5 months in regions of Germany (Bavaria, Saxony, Lower Saxony), UK (Oxford), Poland (Warsaw), Belgium (Leuven), and Sweden (Region Skåne). Seven clinical centers will screen around 330 000 infants. Using a genetic score based on 46 T1D susceptibility single-nucleotide polymorphisms (SNPs) or three SNPS and a first-degree family history for T1D, infants with a high (>10%) genetic risk for developing multiple beta-cell autoantibodies by the age of 6 years are identified. Screening from October 2017 to December 2018 was performed in 50 669 infants. The prevalence of high genetic risk for T1D in these infants was 1.1%. Infants with high genetic risk for T1D are followed up and offered to participate in a randomized controlled trial aiming to prevent beta-cell autoimmunity and T1D by tolerance induction with oral insulin. The GPPAD-02 study provides a unique path to primary prevention of beta-cell autoimmunity in the general population. The eventual benefit to the community, if successful, will be a reduction in the number of children developing beta-cell autoimmunity and T1D.status: publishe