Project description:On deletion of the gene encoding the constant region of the T cell antigen receptor (TCR)alpha chain in mature T cells by induced Cre-mediated recombination, the cells lose most of their TCR from the cell surface within 7--10 days, but minute amounts of surface-bound TCR beta chains are retained for long periods of time. In a situation in which cellular influx from the thymus is blocked, TCR-deficient naive T cells decay over time, the decay rates being faster for CD8(+) cells (t(1/2) approximately 16 days) than for CD4(+) cells (t(1/2) approximately 46 days). TCR(+) naïve cells are either maintained (CD8(+)) or decay more slowly (CD4(+); t(1/2) approximately 78 days.) Numbers of TCR-deficient memory T cells decline very slowly (CD8(+) cells; t(1/2) approximately 52 days) or not at all (CD4(+) cells), but at the population level, these cells fail to expand as their TCR(+) counterparts do. Together with earlier data on T cell maintenance in environments lacking appropriate major histocompatibility complex antigens, these data argue against the possibility that spontaneous ligand-independent signaling by the alpha beta TCR contributes significantly to T-cell homeostasis.
Project description:The origin and function of human double negative (DN) TCR-alpha/beta T cells is unknown. They are thought to contribute to the pathogenesis of systemic lupus erythematosus because they expand and accumulate in inflamed organs. Here we provide evidence that human TCR-alpha/beta CD4- CD8- DN T cells derive exclusively from activated CD8+ T cells. Freshly isolated TCR-alpha/beta DN T cells display a distinct gene expression and cytokine production profile. DN cells isolated from peripheral blood as well as DN cells derived in vitro from CD8+ T cells, produce a defined array of pro-inflammatory mediators that includes IL-1, IL-17, IFN-gama, CXCL3, and CXCL2. These results indicate that, upon activation, CD8+ T cells have the capacity to acquire a distinct phenotype that grants them inflammatory capacity. TCR-alpha-beta+ CD25- T cells from healthy human individuals were sorted into CD4+, CD8+, and CD4-CD8- T cells. Cell lysis and RNA extraction was performed immediately. RNA from each cell subset was pooled.
Project description:Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of alpha beta T cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-kappaB (I kappa B). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts alpha beta T cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.
Project description:In murine T cell development, early thymocytes that productively rearrange the T cell receptor (TCR) beta locus are selected to continue maturation, before TCR alpha expression, by means of a pre-TCR alpha- (pT alpha-) TCR beta heterodimer (pre-TCR). The aim of this study was to identify equivalent stages in human thymocyte development. We show here that variable-diversity-joining region TCR beta rearrangement and the expression of full-length TCR beta transcripts have been initiated in some immature thymocytes at the TCR alpha/beta- CD4+CD8- stage, and become common in a downstream subset of TCR alpha/beta- CD4+CD8+ thymocytes that is highly enriched in large cycling cells. TCR beta chain expression was hardly detected in TCR alpha/beta- CD4+CD8- thymocytes, whereas cytoplasmic TCR beta chain was found in virtually all TCR alpha/beta- CD4+CD8+ blasts. In addition, a TCR beta complex distinct from the mature TCR alpha/beta heterodimer was immunoprecipitated only from the latter subset. cDNA derived from TCR alpha/beta- CD4+CD8+ blasts allowed us to identify and clone the gene encoding the human pT alpha chain, and to examine its expression at different stages of thymocyte development. Our results show that high pT alpha transcription occurs only in CD4+CD8- and CD4+CD8+ TCR alpha/beta- thymocytes, whereas it is weaker in earlier and later stages of development. Based on these results, we propose that the transition from TCR alpha/beta- CD4+CD8- to TCR alpha/beta- CD4+CD8+ thymocytes represents a critical developmental stage at which the successful expression of TCR beta promotes the clonal expansion and further maturation of human thymocytes, independent of TCR alpha.
Project description:The origin and function of human double negative (DN) TCR-alpha/beta T cells is unknown. They are thought to contribute to the pathogenesis of systemic lupus erythematosus because they expand and accumulate in inflamed organs. Here we provide evidence that human TCR-alpha/beta CD4- CD8- DN T cells derive exclusively from activated CD8+ T cells. Freshly isolated TCR-alpha/beta DN T cells display a distinct gene expression and cytokine production profile. DN cells isolated from peripheral blood as well as DN cells derived in vitro from CD8+ T cells, produce a defined array of pro-inflammatory mediators that includes IL-1, IL-17, IFN-gama, CXCL3, and CXCL2. These results indicate that, upon activation, CD8+ T cells have the capacity to acquire a distinct phenotype that grants them inflammatory capacity.
Project description:New biomarkers with improved accuracy could be helpful for monitoring disease in patients with Non-Hodgkin's lymphomas (NHL). Towards this end, we have explored the feasibility of identifying the sequence of rearranged IgH genes using next-generation sequencing, then using PCR to detect specific rearranged DNA fragments in patients' plasma. By capturing and sequencing the IgH genomic regions (IgCap), we were able to detect and precisely determine the sequence of rearranged IgH loci in the tumors of three NHL patients. Moreover, circulating rearranged DNA fragments could be identified in the plasma of all three patients. Even in cases wherein tumor biopsies were unavailable, we were able to use the IgH capture approach to identify rearranged DNA loci in the plasma of 8 of 14 patients. IgCap may enable a more informed management of selected patients with NHL and other B-cell malignancies in the future.
Project description:Despite being a major component of Lewy bodies and Lewy neurites, pathogenic variants in the gene encoding alpha-Synuclein (α-Syn) are rare. To date, only four missense variants in the SNCA gene, encoding α-Syn have unequivocally been shown to be disease-causing. We here describe a Parkinson´s disease patient with early cognitive decline carrying an as yet not fully characterized variant in SNCA (NM_001146055: c.44T > C, p.V15A). We used different cellular models, including stably transfected neuroblastoma (SH-SY5Y) cell cultures, induced pluripotent stem cell (iPSC)-derived neuronal cultures, and generated a Drosophila model to elucidate the impact of the p.V15A variant on α-Syn function and aggregation properties compared to other known pathogenic variants. We demonstrate that p.V15A increased the aggregation potential of α-Syn and the levels of apoptotic markers, and impaired the mitochondrial network. Moreover, p.V15A affects the flying ability and survival of mutant flies. Thus, we provide supporting evidence for the pathogenicity of the p.V15A variant, suggesting its inclusion in genetic testing approaches.
Project description:IKZF1 encodes Ikaros, a zinc finger-containing transcription factor crucial to the development of the hematopoietic system. Germline pathogenic variants in IKZF1 have been reported in patients with acute lymphocytic leukemia and immunodeficiency syndromes. Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by erythroid hypoplasia, associated with a spectrum of congenital anomalies and an elevated risk of certain cancers. DBA is usually caused by heterozygous pathogenic variants in genes that function in ribosomal biogenesis; however, in many cases the genetic etiology is unknown. We identified a germline IKZF1 variant, rs757907717 C > T, in identical twins with DBA-like features and autoimmune gastrointestinal disease. rs757907717 C > T results in a p.R381C amino acid change in the IKZF1 Ik-x isoform (p.R423C on isoform Ik-1), which we show is associated with altered global gene expression and perturbation of transcriptional networks involved in hematopoietic system development. These data suggest that this missense substitution caused a DBA-like syndrome in this family because of alterations in hematopoiesis, including dysregulation of networks essential for normal erythropoiesis and the immune system.
Project description:BackgroundGene expression has long been known to be influenced by the relative proximity of DNA regulatory elements. Topologically associating domains (TADs) are self-interacting genomic regions involved in regulating gene expression by controlling the proximity of these elements. Prior studies of TADs and their biological roles have revealed correlations between TAD changes and cellular differentiation. Here, we used Hi-C and RNA-seq data to correlate TCR-induced changes in TAD structure and gene expression in human CD4+ T cells.ResultsWe developed a pipeline, Differentially Expressed Gene Enrichment Finder (DEGEF), that identifies regions of differentially expressed gene enrichment. Using DEGEF, we found that TCR-regulated genes cluster non-uniformly across the genome and that these clusters preferentially localized in regions of TAD rearrangement. Interestingly, clusters of upregulated genes preferentially formed new Hi-C contacts compared to downregulated clusters, suggesting that TCR-activated CD4+ T cells may regulate genes by changing stimulatory contacts rather than inhibitory contacts.ConclusionsOur observations support a significant relationship between TAD rearrangements and changes in local gene expression. These findings indicate potentially important roles for TAD rearrangements in shaping their local regulatory environments and thus driving differential expression of nearby genes during CD4+ T cell activation. Moreover, they provide new insights into global mechanisms that regulate gene expression.
Project description:AimsWe aimed to evaluate the prognostic value of routine use of PCR amplification of immunoglobulin gene rearrangements in bone marrow (BM) staging in patients with follicular lymphoma (FL).MethodsClonal rearrangements were assessed by immunoglobulin heavy and light-chain gene rearrangement analysis in BM aspirates from 96 patients diagnosed with FL and related to morphological detection of BM involvement in biopsies. In 71 patients, results were also compared with concurrent flow cytometry analysis.ResultsBM involvement was detected by PCR in 34.4% (33/96) of patients. The presence of clonal rearrangements by PCR was associated with advanced clinical stage (I-III vs IV; p<0.001), high FL International Prognostic Index (FLIPI) score (0-1, 2 vs ≥3; p=0.003), and detection of BM involvement by morphology and flow cytometry analysis (p<0.001 for both). PCR-positive patients had a significantly poorer survival than PCR-negative patients (p=0.001, log-rank test). Thirteen patients positive by PCR but without morphologically detectable BM involvement, had significantly poorer survival than patients with negative morphology and negative PCR result (p=0.002). The poor survival associated with BM involvement by PCR was independent of the FLIPI score (p=0.007, Cox regression). BM involvement by morphology or flow cytometry did not show a significant impact on survival.ConclusionsOur results showed that routine use of PCR-based clonality analysis significantly improved the prognostic impact of BM staging in patients with FL. BM involvement by PCR was also an independent adverse prognostic factor.