Project description:Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans. Naïve and activated CD4 T cells, CD8 T cells and B cells were compared for their N-linked glycan structures by MALDI-TOF MS profiling and for expression of glycan transferase genes to assess the biosynthetic basis for any change observed. The major change observed in activated CD4 and CD8 T cells was dramatic reduction of sialylated bi-antennary N-glycans carrying the terminal NeuGc?2-6Gal sequence, and corresponding increase in glycans carrying the Gal?1-3Gal sequence. This change was accounted for by a decrease in the expression of the sialyltransferase ST6Gal, and increase in the expression of the galactosyltransferase ?1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases were increased and decreased, respectively. Keywords = N-linked glycosylation, T cell, B cell, activation, glycosyltransferase, carbohydrate, glycomics, glycan, galactosyltransferase, sialyltransferase Keywords: other
Project description:<p>The etiologies of primary immunodeficiencies often yield novel insights about the immune system. Although a genetic etiology has been suspected for patients with abnormally low CD4+ T cells in the absence of HIV infection or any known causes of lymphopenia, no genetic mutation has been described to date for any case of primary CD4 lymphopenia. In this study, we characterized a non-consanguineous family with two non-HIV infected boys exhibiting an inverted CD4 to CD8 T cell ratio and a history of recurrent chronic viral infections since birth. Consistent with a decreased thymic output of CD4+ T cells, the percentage of CD31+ cells in the CD4+ naive population of these patients was decreased. In addition, the activation of T cells was significantly impaired in the patient upon TCR stimulation. Given the mother's T cells show completely skewed X chromosome inactivation, we suspected that the nature of this disease is X-linked. We performed X-chromosome exon-capture targeted single-end Solexa sequencing on two brothers and the mother and found a 10 base pair deletion at an intron-exon junction of Magnesium Transporter 1 (MAGT1), a Mg<sup>2+</sup> specific transporter. We confirmed that this deletion leads to altered splicing, frameshift, early termination of the mRNA, and deficient protein expression in the lymphocytes of the two patients. Moreover, knockdown of this transporter in T cells isolated from healthy donors can recapitulate the observed T cell activation defect while its ectopic expression in the patients' lymphocytes can restore T cell stimulation. Our discovery highlights the significance of this transporter to T cell function.</p>
Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive proliferation of T-lymphocytes usually associated with oncogenic activation of NOTCH1 signaling. Using a bone marrow transplantation approach, we have modeled murine CD4+ CD8+ T-ALL by overexpressing DNMT3A R882H in Tet2-/- multipotent progenitors. T-ALL derived from NOTCH1 L1601PdelP Tet2-/-, NOTCH1 L1601PdelP Tet2+/+ or TCL1A progenitors were used for comparison, as well as normal Tet2+/+ and Tet2-/- CD4+ CD8+ double positive (DP) thymocytes.
Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive proliferation of T-lymphocytes usually associated with oncogenic activation of NOTCH1 signaling. Using a bone marrow transplantation approach, we have modeled murine CD4+ CD8+ T-ALL by overexpressing DNMT3A R882H in Tet2-/- multipotent progenitors. T-ALL derived from NOTCH1 L1601PdelP Tet2-/-, NOTCH1 L1601PdelP Tet2+/+ or TCL1A progenitors were used for comparison, as well as normal Tet2+/+ and Tet2-/- CD4+ CD8+ double positive (DP) thymocytes.
Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive proliferation of T-lymphocytes usually associated with oncogenic activation of NOTCH1 signaling. Using a bone marrow transplantation approach, we have modeled murine CD4+ CD8+ T-ALL by overexpressing DNMT3A R882H in Tet2-/- multipotent progenitors. T-ALL derived from NOTCH1 L1601PdelP Tet2-/-, NOTCH1 L1601PdelP Tet2+/+ or TCL1A progenitors were used for comparison, as well as normal Tet2+/+ and Tet2-/- CD4+ CD8+ double positive (DP) thymocytes.
Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive proliferation of T-lymphocytes usually associated with oncogenic activation of NOTCH1 signaling. Using a bone marrow transplantation approach, we have modeled murine CD4+ CD8+ T-ALL by overexpressing DNMT3A R882H in Tet2-/- multipotent progenitors. T-ALL derived from NOTCH1 L1601PdelP Tet2-/-, NOTCH1 L1601PdelP Tet2+/+ or TCL1A progenitors were used for comparison, as well as normal Tet2+/+ and Tet2-/- CD4+ CD8+ double positive (DP) thymocytes.
Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive proliferation of T-lymphocytes usually associated with oncogenic activation of NOTCH1 signaling. Using a bone marrow transplantation approach, we have modeled murine CD4+ CD8+ T-ALL by overexpressing DNMT3A R882H in Tet2-/- multipotent progenitors. T-ALL derived from NOTCH1 L1601PdelP Tet2-/-, NOTCH1 L1601PdelP Tet2+/+ or TCL1A progenitors were used for comparison, as well as normal Tet2+/+ and Tet2-/- CD4+ CD8+ double positive (DP) thymocytes.
Project description:High-resolution quantitative mass-spectrometry reveals similarities and disparities in how murine naïve CD4 and CD8 T cells respond to immune activation and re-structure proteomes as they differentiate to effector cells. The data map core transcriptional, metabolic and protein synthesis machinery, nutrient transporters and environment sensing molecules and reveal differences in the biosynthetic capacity of CD4 and CD8 T cells. One key nutrient sensing kinase is mammalian target of rapamycin complex1 (mTORC1). The current study identifies common and distinct mTORC1 regulated processes and divergent outcomes of mTORC1 inhibition in naïve versus effector CD4 and CD8 T cell populations. The data provide a resource that maps how immune activation and mTORC1 reshape CD4 and CD8 T cell proteomes and highlights that a deep understanding of T cell phenotype requires modelling of the impact of immune regulators on protein copy number, cellular protein concentrations and the subunit stoichiometry of key protein complexes.
Project description:Dr. Paulson's group investigates the roles of carbohydrate-binding proteins involved in immune regulation and human disease, through their interaction with carbohydrate groups expressed on cell surface glycoconjugates. This laboratory is currently focused on how sialoside ligands modulate Siglec functions and on understanding the molecular basis of glycosylation changes following differentiation and activation of leukocytes. Evaluation of Glycosyltransferases and CBP expression in B cells, CD4+ and CD8+ T cells following activation. The overall goal of this study is to understand the molecular regulation of cell surface glycosylation in mouse lymphocytes before and after activation. Initial studies using lectins and antibodies showed differences in glycan expression following activation, which included a marked increase in PNA binding. Interestingly, this increase was observed in B, CD4+ and CD8+ cells after 72h of activation. Our aim is to elucidate the molecular basis for such changes in B and T lymphocytes (CD4+ and CD8+), focusing on the mRNA expression of glycogenes before and after activation of mixed spleenocytes. In brief, the experimental protocol is as follows: splenocytes are isolated from C57Bl/6 mice (6-10 wks old) and cultured for 0, 24, 48 and 72 hrs. Cultures containe either anti-CD3/IL2/IL4 or anti-IgM/IL4 to activate T cells and B cells respectively. At each time point, B, CD4+ and CD8+ cells are purified by positive selection using magnetic beads and total RNA is prepared from each cell type.
Project description:The purpose of this microarray study is to characterize molecular and functional differences between CD4+CD300a+ vs CD4+CD300a- T lymphocytes, and between CD8+CD300a+ vs CD8+CD300a- T lymphocytes, respectively.