Functional Interrogation of Primary Human T Cells via CRISPR Genetic Editing.

Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions. Toward this goal, we used lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs that targeted either coding or noncoding regions of genes of interest. We showed that multiple genes (CD4, CD45, CD95) could be simultaneously and stably deleted in naive, memory, effector, or regulatory T cell (Treg) subsets at very high efficiency. Additionally, nuclease-deficient Cas9, associated with a transcriptional activator or repressor, can downregulate or increase expression of genes in T cells. For example, expression of glycoprotein A repetitions predominant (GARP), a gene that is normally and exclusively expressed on activated Tregs, could be induced on non-Treg effector T cells by nuclease-deficient Cas9 fused to transcriptional activators. Further analysis determined that this approach could be used in mapping promoter sequences involved in gene transcription. Through this CRISPR/Cas9-mediated genetic editing we also demonstrated the feasibility of human T cell functional analysis in several examples: 1) CD95 deletion inhibited T cell apoptosis upon reactivation; 2) deletion of ORAI1, a Ca2+ release-activated channel, abolished Ca2+ influx and cytokine secretion, mimicking natural genetic mutations in immune-deficient patients; and 3) transcriptional activation of CD25 or CD127 expression enhanced cytokine signaling by IL-2 or IL-7, respectively. Taken together, application of the CRISPR toolbox to human T cell subsets has important implications for decoding the mechanisms of their functional outputs.

Progesterone receptor membrane component-1 (PGRMC1) and PGRMC-2 interact to suppress entry into the cell cycle in spontaneously immortalized rat granulosa cells.

Progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 are expressed in rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs) but their biological roles are not well defined. The present studies demonstrate that depleting either Pgrmc1 or Pgrmc2 in SIGCs increases entry into the cell cycle but does not increase cell proliferation. Rather, PGRMC1 and/or PGRMC2-deplete cells accumulate in metaphase and undergo apoptosis. Because both PGRMC1 and PGRMC2 localize to the mitotic spindle, their absence likely accounts for cells arresting in metaphase. Moreover, pull-down assays, colocalization studies and in situ proximity ligation assays (PLA) indicate that PGRMC1 binds PGRMC2. Disrupting the PGRMC1:PGRMC2 complex through the use of siRNA or the cytoplasmic delivery of a PGRMC2 antibody increases entry into the cell cycle. Conversely, overexpressing either PGRMC1-GFP or GFP-PGRMC2 fusion protein inhibits entry into the cell cycle. Subsequent studies reveal that depleting PGRMC1 and/or PGRMC2 reduces the percentage of cells in G0 and increases the percentage of cells in G1. These observations indicate that in addition to their role at metaphase, PGRMC1 and PGRMC2 are involved in regulating entry into the G1 stage of the cell cycle. Interestingly, both PGRMC1 and PGRMC2 bind GTPase-activating protein-binding protein 2 (G3BP2) as demonstrated by pull-down assays, colocalization assays, and PLAs. G3bp2 siRNA treatment also promotes entry into the G1 stage. This implies that dynamic changes in the interaction among PGRMC1, PGRMC2, and G3BP2 play an important protein regulating the rate at which SIGCs enter into the cell cycle.

Tuning of human MAIT cell activation by commensal bacteria species and MR1-dependent T-cell presentation.

Human mucosal-associated invariant T (MAIT) cell receptors (TCRs) recognize bacterial riboflavin pathway metabolites through the MHC class 1-related molecule MR1. However, it is unclear whether MAIT cells discriminate between many species of the human microbiota. To address this, we developed an in vitro functional assay through human T cells engineered for MAIT-TCRs (eMAIT-TCRs) stimulated by MR1-expressing antigen-presenting cells (APCs). We then screened 47 microbiota-associated bacterial species from different phyla for their eMAIT-TCR stimulatory capacities. Only bacterial species that encoded the riboflavin pathway were stimulatory for MAIT-TCRs. Most species that were high stimulators belonged to Bacteroidetes and Proteobacteria phyla, whereas low/non-stimulator species were primarily Actinobacteria or Firmicutes. Activation of MAIT cells by high- vs low-stimulating bacteria also correlated with the level of riboflavin they secreted or after bacterial infection of macrophages. Remarkably, we found that human T-cell subsets can also present riboflavin metabolites to MAIT cells in a MR1-restricted fashion. This T-T cell-mediated signaling also induced IFNγ, TNF and granzyme B from MAIT cells, albeit at lower level than professional APC. These findings suggest that MAIT cells can discriminate and categorize complex human microbiota through computation of TCR signals depending on antigen load and presenting cells, and fine-tune their functional responses.

julius seizure, a Drosophila Mutant, Defines a Neuronal Population Underlying Epileptogenesis.

Epilepsy is a neural disorder characterized by recurrent seizures. Bang-sensitive Drosophila represent an important model for studying epilepsy and neuronal excitability. Previous work identified the bang-sensitive gene slamdance (sda) as an allele of the aminopeptidase N gene. Here we show through extensive genetic analysis, including recombination frequency, deficiency mapping, transposon insertion complementation testing, RNA interference (RNAi), and genetic rescue that the gene responsible for the seizure sensitivity is julius seizure (jus), formerly CG14509, which encodes a novel transmembrane domain protein. We also describe more severe genetic alleles of jus RNAi-mediated knockdown of jus revealed that it is required only in neurons and not glia, and that partial bang-sensitivity is caused by knockdown in GABAergic or cholinergic but not glutamatergic neurons. RNAi knockdown of jus at the early pupal stages leads to strong seizures in adult animals, implicating that stage as critical for epileptogenesis. A C-terminal-tagged version of Jus was generated from a fosmid genomic clone. This fosmid fusion rescued the bang-sensitive phenotype and was expressed in the optic lobes and the subesophageal and thoracic abdominal ganglia. The protein was primarily localized in axons, especially in the neck connectives, extending into the thoracic abdominal ganglion.