Forward genetic analysis of memory CD8+ T lymphocyte development and long-term maintenance by in vivo chemical germline mutagenesis

The establishment and long-term maintenance of virus-specific memory CD8+ cytotoxic T lymphocytes (CTL) is genetically controlled. However, not all genes involved in this process have been identified in vivo to date and their identity and function have to be clarified. To identify novel genes which regulate the development of memory cells, we have undertaken an in vivo forward genetic approach based on germline mutagenesis screening. Random point mutations were introduced in the genome of wild-type mice by N-ethyl-N-nitrosourea (ENU) treatment. A library of third generation mutants was then screened for genetic defects affecting their immune responses to Lymphocytic Choriomeningitis virus (LCMV). Altered immune responses to LCMV in mutant mice were detected by measuring the percentage of antigen-specific CTL, their cell death and the composition of the LCMVspecific CD8+ T cell pool at the peak of expansion and at the beginning of the memory phase. Any individuals with outstanding cell population numbers or characteristics were isolated as phenodeviants. Once a mutation was confirmed to affect the germline, a stable strain was established and the mutation was mapped by positional cloning. Through this process, we have isolated 3 mutants: memi, binu and alois. Our first mutant, memi was isolated based on its very low CD8+ T cell memory population following LCMV infections. Using single nucleotide polymorphism linkage analysis, we identified memi as a loss-of-function mutation in the gene encoding deoxycytidine kinase (dCK), a crucial enzyme of the nucleoside salvage pathway. In addition to the immunological phenotype observed after viral infection, we have characterized significant changes in T and B lymphocyte development and their cell-intrinsic/extrinsic peripheral homeostasis in naive memi mice. This mutant is valuable especially to understand how a defect in nucleoside salvage impacts on lymphocyte development and behaviour. The other two mutants, binu and alois, which present increased and decreased CD8+ T memory precursors upon LCMV infection, respectively, are currently being mapped. To our knowledge, no mutant with exactly the same phenotypes as binu or alois has been described to date and we therefore believe that they have strong potential to be novel mutants. Overall, our ENU mutagenesis was successful both in terms of the number of mutants we have isolated and the types of mutations they represent, affecting known and potential new immune regulators

Clinical and genetic data of the 4 heart samples with potentially disease-causing <i>OBSCN</i> mutations.

<p>The locations of the mutations in the Obscurin B domain diagram are shown, based on the model of Ackerman et al. 2014 (ref 14) (Fig E in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138568#pone.0138568.s001" target="_blank">S1 File</a>)</p

Immunofluorescence images of donor and <i>OBSCN</i> mutation- containing human heart tissue sections labelled with Ob59 antibody (green) and anti- α-actinin or anti-myomesin (red).

<p>The monochrome panels show the obscurin immunofluorescence on its own.</p

Analysis of obscurin protein expression in tissue samples.

<p>Left: Identification of obscurin in cardiac myofibrils by 2% agarose/2% PAA/SDS gel electrophoresis. Left, SYPRO Ruby protein stain, Right Western blot Ob59 antibody. Centre: Obscurin expression in myofibrils. Western blot of 4–18% gradient SDS-PAGE. Left: membrane stained with MemCode protein stain, right: probed with Ob59 antibody. For quantification obscurin was normalised to α-actinin. Right: Identification of obscurin in whole heart muscle homogenates. Top, MemCode protein stain; bottom, Western blot with Ob59 antibody.</p

Quantification of Obscurin content of human cardiac myofibrils.

<p>A. 4–18% SDS-PAGE of muscle myofibril fraction. Western blot reversibly stained for total protein by MemCode and then probed with Obscurin antibody Ob59. Obscurin band is normalized to the α-actinin band as a loading control and further normalized to D21, present in every slab gel to control for inter-gel variation. B. Relative obscurin protein level normalised to D21. D4, D14, and D20 are compared with 7 DCM samples without obscurin mutations, 6 donor heart samples and 3 myectomy samples (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138568#pone.0138568.s001" target="_blank">S1 File</a> Figs A, B). Data from 22 slab gels similar to that in 2A pooled. The bars represent sem for replicate measurements on the same sample.</p

<i>ZBTB17 </i>(<i>MIZ1</i>) Is Important for the Cardiac Stress Response and a Novel Candidate Gene for Cardiomyopathy and Heart Failure

BACKGROUND: Mutations in sarcomeric and cytoskeletal proteins are a major cause of hereditary cardiomyopathies, but our knowledge remains incomplete as to how the genetic defects execute their effects. METHODS AND RESULTS: We used cysteine and glycine-rich protein 3 (CSRP3), a known cardiomyopathy gene, in a yeast two-hybrid screen and identified zinc finger and BTB domain containing protein 17 (ZBTB17) as a novel interacting partner. ZBTB17 is a transcription factor that contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus. ZBTB17 expression protected cardiac myocytes from apoptosis in vitro and in a mouse model with cardiac myocyte-specific deletion of Zbtb17, which develops cardiomyopathy and fibrosis after biomechanical stress. ZBTB17 also regulated cardiac myocyte hypertrophy in vitro and in vivo in a calcineurin-dependent manner. CONCLUSIONS: We revealed new functions for ZBTB17 in the heart, a transcription factor which may play a role as a novel cardiomyopathy gene