Project description:The aim of the dataset was to study on a genome-wide level the impact of Lat deficiency on gene expression in resting and activated CD4+ T cells Lat+ and Lat? CD4+ T cells were isolated from lymph nodes and spleen of Latfl-dtr Tmat-Cre mice using a Dynabeads untouched mouse CD4 cells kit (Life Technology) and further purified by cell sorting. Lat+ CD4+ T cells were defined as: CD5+, hDTR+, CD8?, TCR???, CD25?, CD69?, CD62L+, lineage (CD11c, CD11b, CD19, CD45R, CD161)? and Lat? CD4+ Tcells were defined as: CD5+, hDTR?, CD8?, TCR???, CD25?, CD69?, CD62L+, lineage (CD11c, CD11b, CD19, CD45R, CD161)?. Sorted Lat+ or Lat? CD4+ T cells were then kept in vitro for 4 hours without stimulation or activated for 4 hours using anti-CD3 antibody and anti-CD28 antibody. Cell samples corresponding to three biological replicates were analyzed and gene expression profiles were obtained from total RNA.
Project description:Transmembrane adaptors on T cells play crucial roles in TCR signal transduction. Although the TCR-LAT signal-transduction axis occupies a central place in T cell activation, it does not work in isolation and is tuned by other T-cell surface receptors among which stand the CD5 and CD6 receptors. Here we assessed the extent of transcriptional changes that initiated by TCR-CD28 pathways in the absence of CD5, of CD6, of both CD5 and CD6, or of LAT by RNA-seq analysis.
Project description:T cell antigen receptor (TCR) signaling depends upon the kinases Lck and Zap70. Lck phosphorylates the TCR, facilitating Zap70 recruitment to the stimulated TCR. Lck also phosphorylates Zap70, relieving its auto-inhibition and activating its catalytic domain. Zap70 then phosphorylates the critical adaptors LAT and SLP76 which serve to nucleate key effector molecules required for downstream responses. However, mechanisms facilitating the interaction of Zap70 with its substrates have not been described. We report an evolutionarily conserved proline-rich motif in LAT is important for Zap70-induced phosphorylation of LAT and downstream signaling. This LAT proline-rich motif associated with the Lck SH3 domain, thereby facilitating Zap70-mediated phosphorylation of LAT and downstream functions. Our results suggest Lck orchestrates multiple steps in TCR signaling including the newly described facilitation of the interaction of Zap70 with its substrate LAT. This previously unrecognized feature of TCR proximal signaling may contribute to the development of more immunomodulatory therapies.
Project description:Chimeric antigen receptor (CAR) T cell therapy have demonstrated remarkable success in treating B-cell malignancies that are refractory to standard therapies; however, preclinical and clinical studies have demonstrated that CAR T cell efficacy is greatly reduced against antigen-low tumors. This was exemplified in a clinical trial of CD22-directed CAR T cells where post-CAR relapses were driven by a decrease in the surface expression of the CD22 antigen. To address the poor response of CAR T cells to antigen-low tumor cells, we performed global phosphoproteomics using a SILAC/mass spectrometry approach to examine signal transduction events within CAR T cells in response to high- and low-levels of CD22 antigen. Stimulation with low levels of CD22 antigen resulted in decreased activation of several canonical T cell signaling pathways in CAR T cells, suggesting poor utilization of LAT. To overcome this inefficiency of LAT activation, we designed a bicistronic construct consisting of a clinically active 2nd generation CD22-BBz CAR along with a novel Adjunctive LAT Activating CAR incorporating the intracellular signaling domain of LAT (ALA-CART). ALA-CART cells demonstrated enhanced phosphorylation of LAT and restored downstream signaling in response to low levels of CD22. In xenograft models, ALA-CART cells completely eradicated CD22-low leukemia, significantly extending survival of mice in comparison to the clinically-active, standard CD22-BBz CAR T cells. Compared to the standard CD22-BBz CAR T cells, ALA-CART cells exhibited transcriptional differences in the resting state reflecting a less differentiated state, which corresponded to an enrichment of T stem cell memory cells and enhanced in vivo persistence. Thus, through the identification of CAR signaling deficits, we rationally designed the ALA-CART platform which improves the sensitivity and persistence of CAR T cells to target antigen-low cells, overcoming a mechanism of resistance to standard CAR therapies.
2025-02-04 | GSE236468 | GEO
Project description:RNA Sequencing Analysis of Treated J-Lat 5a8 Cells
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.