Citations

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

SummaryRheumatoid arthritis (RA) is associated with the presence of certain HLA class II genes. However, why some individuals carrying RA non‐associated alleles develop arthritis is still unexplained. The trans‐heterodimer between two RA non‐associated HLA genes can render susceptibility to develop arthritis in humanized mice, DQA1*0103/DQB1*0604, suggesting a role for DQ α chains in pathogenesis. In this study we determined the role of DQA1 in arthritis by using mice expressing DQA1*0103 and lacking endogenous class II molecules. Proximity ligation assay showed that DQA1*0103 is expressed on the cell surface as a dimer with CD74. Upon immunization with type II collagen, DQA1*0103 mice generated an antigen‐specific cellular and humoral response and developed severe arthritis. Structural modelling suggests that DQA1*0103/CD74 form a pocket with similarity to the antigen binding pocket. DQA1*0103 mice present type II collagen‐derived peptides that are not presented by an arthritis‐resistant DQA1*0103/DQB1*0601 allele, suggesting that the DQA1*0103/CD74 dimer may result in presentation of unique antigens and susceptibility to develop arthritis. The present data provide a possible explanation by which the DQA1 molecule contributes to susceptibility to develop arthritis.

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.

ABSTRACTBACKGROUNDDisability progression in multiple sclerosis (MS) remains incompletely understood. Unlike lesional measures, central nervous system atrophy has a strong correlation with disability. Theiler's murine encephalomyelitis virus infection in SJL/J mice is an established model of progressive MS. We utilized in vivo MRI to quantify brain and spinal cord atrophy in this model and analyzed the temporal relationship between atrophy and disability.METHODSInfected and control mice were followed for 12 months. Disability was assessed periodically using rotarod assay. Volumetric MRI datasets were acquired at 7 Tesla. Ventricular volume and C4‐5 spinal cord cross‐sectional area measurements were performed using Analyze 10.RESULTSAt 3 months, brain atrophy reached statistical significance (P = .005). In contrast, disability did not differ until 4 months post‐infection (P = .0005). Cord atrophy reached significance by 9 months (P = 0.009). By 12 months, brain atrophy resulted in 111.8% increased ventricular volume (P = .00003), while spinal cord cross‐sectional area was 25.6% reduced (P = .001) among cases.CONCLUSIONSOur results suggest that significant brain atrophy precedes and predicts the development of disability, while spinal cord atrophy occurs late and correlates with severe disability. The observed temporal relationship establishes a framework for mechanisms of disability progression and enables further investigations of their underlying substrate.

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.

Recombinant virus vaccines are often less effective due to immunodominant responses against endogenous vector antigens. However, the use of small RNA virus vectors provides an opportunity to limit host exposure to endogenous virus antigens and focus immune responses on the desired vaccine antigen. Using the Daniel's strain of Theiler's murine encephalomyelitis virus, we have identified strategies to modulate responses to endogenous viral proteins by manipulating the host CD8+ T-cell repertoire prior to infection or through the use of mutations introduced into the virus genome. Both of these approaches enhance responses to vaccine antigens introduced into the picornavirus. However, the use of mutant immunodominant epitopes provides an opportunity for enhancing vaccine responses without further manipulation of the host. Using this strategy, we demonstrate that modification of the consensus MHC class I anchor residue within the virus genome can promote enhanced immunity to foreign antigens and self-antigens embedded in the virus genome.

Abstract Peptide immunotherapy both activates and suppresses the T cell response against known peptide Ags. Although pretreatment with VP2121–130 peptide inhibits the development of antiviral CTL specific for the immunodominant Db:VP2121–130 epitope expressed during acute Theiler’s murine encephalomyelitis virus infection, i.v. injection of this same peptide or MHC tetramers containing the peptide during an ongoing antiviral CTL response results in a peptide-induced fatal syndrome (PIFS) within 48 h. Susceptibility to PIFS is dependent on peptide-specific CD8+ T cells, varies among inbred strains of mice, and is not mediated by traditionally defined mechanisms of shock. Analyses using bone marrow chimeras and mutant mice demonstrate that susceptibility to PIFS is determined by the genotype of bone marrow-derived cells and requires the expression of perforin. Animals responding to peptide treatment with PIFS develop classical stress responses in the brain. These findings raise important considerations for the development of peptide therapies for active diseases to modify immune responses involving expanded populations of T cells. In summary, treatment with peptides or MHC-tetramers during a peptide-specific immune response can result in a fatal shock-like syndrome. Susceptibility to the syndrome is genetically determined, is mediated by CD8+ T cells, and requires expression of perforin. These findings raise concerns about the use of peptides and MHC tetramers in therapeutic schemes.

Abstract Both CD8 and the TCR bind to MHC class I molecules during physiologic T cell activation. It has been shown that for optimal T cell activation to occur, CD8 must be able to bind the same class I molecule that is bound by the TCR. However, no direct evidence for the class I-dependent association of CD8 and the TCR has been demonstrated. Using fluorescence resonance energy transfer, we show directly that a single class I molecule causes TCR/CD8 interaction by serving as a docking molecule for both CD8 and the TCR. Furthermore, we show that CD3ε is brought into close proximity with CD8 upon TCR/CD8 association. These interactions are not dependent on the phosphorylation events characteristic of T cell activation. Thus, MHC class I molecules, by binding to both CD8 and the TCR, mediate the reorganization of T cell membrane components to promote cellular activation.

Abstract Blood–brain barrier (BBB) disruption is a common feature of numerous neurologic disorders. A fundamental question in these diseases is the extent inflammatory immune cells contribute to CNS vascular permeability. We have previously shown that CD8 T cells play a critical role in initiating BBB disruption in the peptide-induced fatal syndrome model developed by our laboratory. However, myelomonocytic cells such as neutrophils have also been implicated in promoting CNS vascular permeability and functional deficit in murine models of neuroinflammatory disease. For this reason, we evaluated neutrophil depletion in a murine model of CD8 T cell-initiated BBB disruption by employing traditionally used anti-granulocyte receptor-1 mAb RB6-8C5 and Ly-6G–specific mAb 1A8. We report that CNS-infiltrating antiviral CD8 T cells express high levels of granulocyte receptor-1 protein and are depleted by treatment with RB6-8C5. Mice treated with RB6-8C5, but not 1A8, display: 1) intact BBB tight junction proteins; 2) reduced CNS vascular permeability visible by gadolinium-enhanced T1-weighted magnetic resonance imaging; and 3) preservation of motor function. These studies demonstrate that traditional methods of neutrophil depletion with RB6-8C5 are broadly immune ablating. Our data also provide evidence that CD8 T cells initiate disruption of BBB tight junction proteins and CNS vascular permeability in the absence of neutrophil support.