Kelcher, April M. Huseby

Quantitative activity through the TCR-proximal protein network specifies thymic positive versus negative selection.

ABSTRACT Brain atrophy is a common feature of numerous neurologic diseases in which the role of neuroinflammation remains ill‐defined. In this study, we evaluated the contribution of major histocompatibility complex class I molecules to brain atrophy in Theiler's murine encephalomyelitis virus (TMEV)–infected transgenic FVB mice that express the D b class I molecule. FVB/D b and wild‐type FVB mice were evaluated for changes in neuroinflammation, virus clearance, neuropathology, and development of brain atrophy via T2‐weighted MRI and subsequent 3‐dimensional volumetric analysis. Significant brain atrophy and hippocampal neuronal loss were observed in TMEV‐infected FVB/D b mice, but not in wild‐type FVB mice. Brain atrophy was observed at 1 mo postinfection and persisted through the 4‐mo observation period. Of importance, virus‐infected FVB/D b mice elicited a strong CD8 T‐cell response toward the immunodominant D b ‐restricted TMEV‐derived peptide, VP2 121–130 , and cleared TMEV from the CNS. In addition, immunofluorescence revealed CD8 T cells near virus‐infected neurons; therefore, we hypothesize that class I restricted CD8 T‐cell responses promote development of brain atrophy. This model provides an opportunity to analyze the contribution of immune cells to brain atrophy in a system where persistent virus infection and demyelination are not factors in long‐term neuropathology.—Huseby Kelcher, A. M., Atanga, P. A., Gamez, J. D., Cumba Garcia, L. M., Teclaw, S. J., Pavelko, K. D., Macura, S. I., Johnson, A. J. Brain atrophy in picornavirus‐infected FVB mice is dependent on the H‐2Db class I molecule. FASEB J. 31, 2267–2275 (2017). www.fasebj.org

Background— A portion of sudden cardiac deaths can be attributed to structural heart diseases, such as hypertrophic cardiomyopathy (HCM) or cardiac channelopathies such as long-QT syndrome (LQTS); however, the underlying molecular mechanisms are distinct. Here, we identify a novel CACNA1C missense mutation with mixed loss-of-function/gain-of-function responsible for a complex phenotype of LQTS, HCM, sudden cardiac death, and congenital heart defects. Methods and Results— Whole exome sequencing in combination with Ingenuity variant analysis was completed on 3 affected individuals and 1 unaffected individual from a large pedigree with concomitant LQTS, HCM, and congenital heart defects and identified a novel CACNA1C mutation, p.Arg518Cys, as the most likely candidate mutation. Mutational analysis of exon 12 of CACNA1C was completed on 5 additional patients with a similar phenotype of LQTS plus a personal or family history of HCM-like phenotypes and identified 2 additional pedigrees with mutations at the same position, p.Arg518Cys/His. Whole cell patch clamp technique was used to assess the electrophysiological effects of the identified mutations in Ca V 1.2 and revealed a complex phenotype, including loss of current density and inactivation in combination with increased window and late current. Conclusions— Through whole exome sequencing and expanded cohort screening, we identified a novel genetic substrate p.Arg518Cys/His-CACNA1C, in patients with a complex phenotype including LQTS, HCM, and congenital heart defects annotated as cardiac-only Timothy syndrome. Our electrophysiological studies, identification of mutations at the same amino acid position in multiple pedigrees, and cosegregation with disease in these pedigrees provide evidence that p.Arg518Cys/His is the pathogenic substrate for the observed phenotype.

KLF10 has recently elicited significant attention as a transcriptional regulator of transforming growth factor-β1 (TGF-β1) signaling in CD4+ T cells. In the current study, we demonstrate a novel role for KLF10 in the regulation of TGF-β receptor II (TGF-βRII) expression with functional relevance in antiviral immune response. Specifically, we show that KLF10-deficient mice have an increased number of effector/memory CD8+ T cells, display higher levels of the T helper type 1 cell-associated transcription factor T-bet, and produce more IFN-γ following in vitro stimulation. In addition, KLF10−/− CD8+ T cells show enhanced proliferation in vitro and homeostatic proliferation in vivo. Freshly isolated CD8+ T cells from the spleen of adult mice express lower levels of surface TGF-βRII (TβRII). Congruently, in vitro activation of KLF10-deficient CD8+ T cells upregulate TGF-βRII to a lesser extent compared with wild-type (WT) CD8+ T cells, which results in attenuated Smad2 phosphorylation following TGF-β1 stimulation compared with WT CD8+ T cells. Moreover, we demonstrate that KLF10 directly binds to the TGF-βRII promoter in T cells, leading to enhanced gene expression. In vivo viral infection with Daniel's strain Theiler's murine encephalomyelitis virus (TMEV) also led to lower expression of TGF-βRII among viral-specific KLF10−/− CD8+ T cells and a higher percentage of IFN-γ-producing CD8+ T cells in the spleen. Collectively, our data reveal a critical role for KLF10 in the transcriptional activation of TGF-βRII in CD8+ T cells. Thus, KLF10 regulation of TGF-βRII in this cell subset may likely play a critical role in viral and tumor immune responses for which the integrity of the TGF-β1/TGF-βRII signaling pathway is crucial.