Project description:Our earlier study demonstrated that when CFSE-labeled LCMV-or Pichinde virus-immune spleen leukocytes were transferred into T cell-deficient hosts, the bona fide virus-specific memory cells underwent relatively limited cell division and were substantially diluted in frequency by other more extensively proliferating cells originating from that donor cell population. We questioned how the slowly dividing population, which contained bona fide memory cells, differed from the rapidly dividing cells, which contained âmemory-likeâ cells. As a preliminary screen we performed a comparative genome-wide microarray analysis of genes expressed on sorted rapidly proliferating (CFSE-low) and slowly proliferating (CFSE-high) CD8 cell populations Experiment Overall Design: 2x10^7 CFSE-labeled, Ly5.1+, LCMV-Immune splenocytes were adoptively transferred into congenic T cell ko hosts. Splenocytes were harvested 12 days post-transfer and stained with anti-CD8 Ab, anti-Ly5.1 Ab and 7AAD. 7AAD negative cells were gated, and CFSE-low (> eight divisions) and CFSE-high (0-6 divisions) cells were sorted by flow cytometry using a FACStarPLUS sorter (BD Bioscience, Mountain View, CA). Total RNA was extracted from both CFSE-low and CFSE-high CD8+Ly5.1+ donor populations to compare rapidly vs. slowly dividing CD8 T cells during acute homeostatic proliferation via Affymetrix gene analysis.
Project description:Our earlier study demonstrated that when CFSE-labeled LCMV-or Pichinde virus-immune spleen leukocytes were transferred into T cell-deficient hosts, the bona fide virus-specific memory cells underwent relatively limited cell division and were substantially diluted in frequency by other more extensively proliferating cells originating from that donor cell population. We questioned how the slowly dividing population, which contained bona fide memory cells, differed from the rapidly dividing cells, which contained “memory-like” cells. As a preliminary screen we performed a comparative genome-wide microarray analysis of genes expressed on sorted rapidly proliferating (CFSE-low) and slowly proliferating (CFSE-high) CD8 cell populations Keywords: Division profile comparison
Project description:How systemic metabolic alterations during acute infections impact immune-cell function remains poorly understood. Hess and colleagues demonstrate that acetate rapidly increases during infections, which drives acetylation of GAPDH in memory CD8+ T cells and thereby catalyzes the rapid recall response. Comparison of mRNA profile of control vs. 3d acetate exposed memory OT-I T cells
Project description:Proteome analysis of embryonic neural progenitor cells, comparing slowly-dividing and rapidly-dividing neural progenitor cells (NPCs). Expression of H2B-GFP was transiently induced in transgenic embryos with a Tet-on system by a single injection of 9TB-Dox at E9.5. Subsequently, NPCs (CD133+CD24– cells) were isolated from the LGE based on the GFP fluorescence intensity at E17.5. The top 10% of NPCs according to H2B-GFP fluorescence intensity (GFP++ cells) were collected as slowly dividing NPCs, whereas the middle 45% to 75% (GFP+ cells) and bottom 10% (GFP– cells) of NPCs were collected as rapidly dividing NPC fractions.
Project description:The intestinal epithelium is continuously regenerated by highly proliferative Lgr5+ intestinal stem cells (ISCs). The existence of a population of quiescent ISCs has been suggested yet its identity and features remain controversial. Here we describe that the expression of the RNA-binding protein Mex3a labels a subpopulation of Lgr5+ cells that divide less frequently and contribute to regenerate all intestinal lineages with slow kinetics. Single cell transcriptomic analysis revealed two classes of Lgr5-high cells, one of them defined by the Mex3a-expression program and by low levels of proliferation genes. Lineage tracing experiments show that large fraction of Mex3a+ cell population is continuously recalled into the rapidly dividing self-renewing ISC pool in homeostatic conditions. Chemotherapy and radiation target preferentially rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, which helps sustain the renewal of the intestinal epithelium during treatment.
Project description:Different subsets of the tRNA pool in human are expressed in different cellular conditions. The “proliferation-tRNAs” are induced upon normal and cancerous cell division, while the “differentiation tRNAs” are active in non-dividing, differentiated cells. Here we examine the essentiality of the various tRNAs upon cellular growth and arrest. We established a CRISPR-based editing procedure with sgRNAs that each target a tRNA family. We measured tRNA essentiality for cellular growth and found that most proliferation tRNAs are essential compared to differentiation tRNAs in rapidly growing cell lines. Yet in more slowly dividing lines, the differentiation tRNAs were more essential. In addition, we measured the essentiality of each tRNA family upon response to cell cycle arresting signals. Here we detected a more complex behavior with both proliferation-tRNAs and differentiation-tRNAs showing various levels of essentiality. These results provide the so-far most comprehensive functional characterization of human tRNAs with intricate roles in various cellular states.
Project description:Different subsets of the tRNA pool in human are expressed in different cellular conditions. The “proliferation-tRNAs” are induced upon normal and cancerous cell division, while the “differentiation tRNAs” are active in non-dividing, differentiated cells. Here we examine the essentiality of the various tRNAs upon cellular growth and arrest. We established a CRISPR-based editing procedure with sgRNAs that each target a tRNA family. We measured tRNA essentiality for cellular growth and found that most proliferation tRNAs are essential compared to differentiation tRNAs in rapidly growing cell lines. Yet in more slowly dividing lines, the differentiation tRNAs were more essential. In addition, we measured the essentiality of each tRNA family upon response to cell cycle arresting signals. Here we detected a more complex behavior with both proliferation-tRNAs and differentiation-tRNAs showing various levels of essentiality. These results provide the so-far most comprehensive functional characterization of human tRNAs with intricate roles in various cellular states.
Project description:Different subsets of the tRNA pool in human are expressed in different cellular conditions. The “proliferation-tRNAs” are induced upon normal and cancerous cell division, while the “differentiation tRNAs” are active in non-dividing, differentiated cells. Here we examine the essentiality of the various tRNAs upon cellular growth and arrest. We established a CRISPR-based editing procedure with sgRNAs that each target a tRNA family. We measured tRNA essentiality for cellular growth and found that most proliferation tRNAs are essential compared to differentiation tRNAs in rapidly growing cell lines. Yet in more slowly dividing lines, the differentiation tRNAs were more essential. In addition, we measured the essentiality of each tRNA family upon response to cell cycle arresting signals. Here we detected a more complex behavior with both proliferation-tRNAs and differentiation-tRNAs showing various levels of essentiality. These results provide the so-far most comprehensive functional characterization of human tRNAs with intricate roles in various cellular states.
Project description:Maintenance of memory CD8 T cell effector function is coupled to stability ofeffector DNA methylation programs during homeostatic proliferation