CD8 and CD4 lymphocytes control cytomegalovirus (CMV) infection in immunocompetent individuals, while patients with defective cellular immunity are prone to endogenous reactivation of latent CMV or, like seronegative subjects, prone to primary infection. Administration of CMV-specific CD8 lymphocytes was beneficial for immunocompromised hemopoietic stem cell (HSC) graft recipients. Since CD4 cells contribute to expansion of cytotoxic T lymphocytes (CTL), we defined new T(h) peptides on the immunodominant protein pp65 recognized by CD4 cells from HLA-typed subjects, in the perspective of complementing CTL administration with CMV-specific T(h) cells. Screening by ELISPOT on CD4 and CD8 subsets using overlapping peptides identified 10 novel CD4 peptides. To simplify procedures to generate T cell lines, we used a CD4 peptide library for T cell stimulation instead of ill-defined viral lysates, without the requirement of dendritic cells. This library stimulated CMV-specific CD4 cells. In fact, peptide-induced CD4 cells responded to pp65 and to the viral lysate. These cells were also devoid of alloreactivity after one stimulation cycle. Since Good Manufacturing Procedure-grade peptides can be synthesized, culture conditions are simplified and alloreactivity is rapidly lost, these procedures based on peptide stimulation can facilitate implementation of adoptive reconstitution of CD4 responses in immunocompromised patients also in the case when the HSC allodonor is available for generation of the T cell line.

Recent evidence indicates that the miRNA biogenesis factors DROSHA, DGCR8, and DICER exert non-overlapping functions, and have also roles in miRNA-independent regulatory mechanisms. However, it is currently unknown whether miRNA-independent functions of DGCR8 play any role in the maintenance of neuronal progenitors and during corticogenesis. Here, by phenotypic comparison of cortices from conditional and knockout mice, we show that deletion, in contrast to depletion, leads to premature differentiation of neural progenitor cells and overproduction of TBR1-positive neurons. Remarkably, depletion of miRNAs upon DCGR8 loss is reduced compared to DICER loss, indicating that these phenotypic differences are mediated by miRNA-independent functions of DGCR8. We show that mutations induce an earlier and stronger phenotype in the developing nervous system compared to mutants and that miRNA-independent functions of DGCR8 are critical for corticogenesis. Finally, our data also suggest that the Microprocessor complex, with DROSHA and DGCR8 as core components, directly regulates the transcript, containing evolutionarily conserved hairpins that resemble miRNA precursors, independently of miRNAs.

The development of adequate model systems to study human malignancies is crucial for basic and preclinical research. Here, we exploit the ''immune-privileged'' developmental time window to achieve orthotopic xenotransplantation of human brain tumor cells in wild-type (WT) mice. We find that, when transplanted in utero, human glioblastoma (GBM) cells readily integrate in the embryonic mouse brain mirroring key tumor-associated pathological features such as infiltration, vascularization, and complex tumor microenvironment including reactive astrocytes and host immune cell infiltration. Remarkably, activation of the host IBA1 tumor-associated microglia/macrophages depends on the type of glioma cell transplanted, suggesting our approach allows one to study human GBM interactions with the immune system of WT host mice. The embryonic engraftment model complements existing ones, providing a rapid and valuable alternative to study fundamental biology of human brain tumors in immune competent mice.

No abstract

This procedure can be applied to improve sterility under GMP conditions when T-cell lines are generated for adoptive immunotherapy and may increase biosafety for the staff when cell lines are generated from subjects infected with dangerous pathogens.

Adult neural progenitor cells (aNPCs) ensure lifelong neurogenesis in the mammalian hippocampus. Proper regulation of aNPC fate has thus important implications for brain plasticity and healthy aging. Piwi proteins and the small noncoding RNAs interacting with them (piRNAs) have been proposed to control memory and anxiety, but the mechanism remains elusive. Here, we show that Piwil2 (Mili) is essential for proper neurogenesis in the postnatal mouse hippocampus. RNA sequencing of aNPCs and their differentiated progeny reveal that Mili and piRNAs are dynamically expressed in neurogenesis. Depletion of Mili and piRNAs in the adult hippocampus impairs aNPC differentiation toward a neural fate, induces senescence, and generates reactive glia. Transcripts modulated upon Mili depletion bear sequences complementary or homologous to piRNAs and include repetitive elements and mRNAs encoding essential proteins for proper neurogenesis. Our results provide evidence of a critical role for Mili in maintaining fitness and proper fate of aNPCs, underpinning a possible involvement of the piRNA pathway in brain plasticity and successful aging.

In specific niches of the adult mammalian brain, neural progenitor cells (aNPCs) ensure lifelong neurogenesis. Proper regulation of this process entails important implications for brain plasticity and health. We report that Piwil2 (Mili) and PIWI-interacting RNAs (piRNAs) are abundantly expressed in aNPCs but depleted in their progeny in the adult mouse hippocampus. Loss of function of the piRNA pathway in aNPCs inhibited neurogenesis and increased reactive gliogenesis in vivo and in vitro. PiRNA pathway depletion in cultured aNPCs increased levels of 5S ribosomal RNA, transfer RNAs and mRNAs encoding regulators of translation, resulting in higher polyribosome density and protein synthesis upon differentiation. We propose that the piRNA pathway sustains adult neurogenesis by repressing translation in aNPCs.One sentence summaryThe piRNA pathway is enriched in neural precursors and essential for appropriate neurogenesis by modulating translation

TTF-1/NKX2.1, also known as T/EBP, is a homeodomain-containing gene involved in the organogenesis of the thyroid gland, lung and ventral forebrain. We have already reported that in 3T3 cells, TTF-1/NKX2.1 up-regulates the transcription of nestin, an intermediate filament protein expressed in multipotent neuroepithelial cells, by direct DNA-binding to a HRE/CRE-like site (NestBS) within a CNS-specific enhancer. Here, we demonstrate that TTF-1/NKX2.1 is coexpressed with nestin in the embryonal forebrain. We also performed a transgenic mouse embryo analysis in which NestBS was replaced by the canonical TTF-1/NKX2.1 consensus DNA-binding site (as identified in many thyroid-and lung-specific genes and very divergent from NestBS) or a random mutation. We observed β β β β β-galactosidase expression in forebrain regions where TTF-1/ NKX2.1 is expressed in wild-type embryos, and -to a minor extent-in rostralmost telencephalic regions and thalamus, whereas no β β β β β-galactosidase expression was detected in forebrains of embryos bearing the random mutation. These data show that TTF-1/NKX2.1 regulates the transcription of the nestin gene in vivo through the NestBS site, suggesting that nestin might be at least one of the effectors of TTF-1/NKX2.1 during forebrain development. Finally, we have shown that the transactivating effect of TTF-1/NKX2.1 on the CNS-specific enhancer is unaffected by Retinoic Acid Receptor-α α α α α.