B cell deficiency and anaemia caused by mutations in the murine atp11c gene

Organization of the plasma membrane into specialised substructures in lymphocytes facilitates important biological functions including the initiation of crucial intracellular signalling cascades at the plasma membrane. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. Membrane-bound lipid transporters are believed to generate and dynamically maintain the lipid asymmetry between the two leaflets of the cell membranes, but not much is known about the role of these transporters in a variety of biological systems in mammals. This thesis examines the effect of two ENU-induced mutations of the murine Atp11c gene, which encodes a member of the P4-type ATPase family thought to serve as 'flippases' that mediate the translocation of specific aminophospholipids to the cytoplasmic leaflet of cell membranes. Loss of ATP11C in mice led to a severe B cell deficiency due to a developmental arrest at the pro-B cell stage during early B cell development in the bone marrow. The number of splenic follicular B cells and peritoneal B1 cells was also severely reduced in mutant mice. However, marginal zone B cells as well as other haematopoietic lineages including T, NK and myeloid cells appeared to accumulate normally in mutant mice. Moreover, the requirement for ATP11C in B cells was cell autonomous, and could not be corrected by the expression of pre-rearranged immunoglobulin transgenes or enforced expression of the pro-survival protein BCL-2 or by transgenic expression of IL-7. Further analysis of mutant mice revealed that while the IL-7R-mediated signalling pathway appears mostly intact, the ATP11Camb mutation leads to a defect in the expression and/or signalling through the pre-BCR, which provides essential signals for the development of pre-B cells. In contrast, B cells from B cell receptor (BCR) transgenic ATP11C-deficient mice were able to form germinal centres upon immunisation when adoptively transferred into wild-type host, indicating an intact in vivo signalling through the mature BCR in mutant animals. Functional analysis using fluorescently labelled phospholipid derivatives revealed that cells of the immune system from ATP11C-deficient mice exhibit impaired aminophospholipid flippase activity compared to those from control animals, indicating for the first time that ATP11C functions as a phospholipid flippase in biological membranes. Although the first phospholipid translocase activity was described in erythrocytes about 30 years ago, the characterisation of enzyme activity and its effect on the membrane asymmetry, development and survival of erythrocytes remains to be unveiled. Intriguingly, ATP11C-deficient mice developed anaemia due to a shortened erythrocyte lifespan, exhibited a large proportion of abnormal-shaped erythrocytes, and increased phosphatidylserine exposure on their surface due to impaired flippase activity. Thus, these findings identified ATP11C as a candidate for aminophospholipid translocation activity in erythrocytes. In conclusion, the findings of this thesis provide novel insights into the role of the putative phospholipid transporter ATP11C in B cell development and erythrocyte survival, and suggest a new candidate for inherited B cell deficiency and anaemia in humans

The role of alternative splicing in the control of immune homeostasis and cellular differentiation

Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system

The role of alternative splicing in the control of immune homeostasis and cellular differentiation

Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune syste

Differential Requirement for the CD45 Splicing Regulator hnRNPLL for Accumulation of NKT and Conventional T Cells

Natural killer T (NKT) cells represent an important regulatory T cell subset that develops in the thymus and contains immature (NK1.1lo) and mature (NK1.1hi) cell subsets. Here we show in mice that an inherited mutation in heterogeneous ribonucleoprotein L-like protein (hnRNPLLthunder), that shortens the survival of conventional T cells, has no discernible effect on NKT cell development, homeostasis or effector function. Thus, Hnrpll deficiency effectively increases the NKT∶T cell ratio in the periphery. However, Hnrpll mutation disrupts CD45RA, RB and RC exon silencing of the Ptprc mRNA in both NKT and conventional T cells, and leads to a comparably dramatic shift to high molecular weight CD45 isoforms. In addition, Hnrpll mutation has a cell intrinsic effect on the expression of the developmentally regulated cell surface marker NK1.1 on NKT cells in the thymus and periphery but does not affect cell numbers. Therefore our results highlight both overlapping and divergent roles for hnRNPLL between conventional T cells and NKT cells. In both cell subsets it is required as a trans-acting factor to regulate alternative splicing of the Ptprc mRNA, but it is only required for survival of conventional T cells

ASCT2 (SLC1A5)-Deficient Mice Have Normal B-Cell Development, Proliferation, and Antibody Production

SLC1A5 (solute carrier family 1, member 5) is a small neutral amino acid exchanger that is upregulated in rapidly proliferating lymphocytes but also in many primary human cancers. Furthermore, cancer cell lines have been shown to require SLC1A5 for their survival in vitro. One of SLC1A5's primary substrates is the immunomodulatory amino acid glutamine, which plays an important role in multiple key processes, such as energy supply, macromolecular synthesis, nucleotide biosynthesis, redox homeostasis, and resistance against oxidative stress. These processes are also essential to immune cells, including neutrophils, macrophages, B and T lymphocytes. We show here that mice with a stop codon in Slc1a5 have reduced glutamine uptake in activated lymphocytes and primary fibroblasts. B and T cell populations and maturation in resting mice were not affected by absence of SLC1A5. Antibody production in resting and immunized mice and the germinal center response to immunization were also found to be normal. SLC1A5 has been recently described as a novel target for the treatment of a variety of cancers, and our results indicate that inhibition of SLC1A5 in cancer therapy may be tolerated well by the immune system of cancer patients.This work was supported by Australian National Health and Medical Research Council Grant 1105857 (SB) and Career Development Fellowship 1035858 (AE

The impact of the earlySTEM program on teacher and student outcomes: The role of teachers’ involvement in the program development.

This research aimed to investigate and compare teachers‘ conceptualizations of their students‘ and their own outcomes of our earlySTEM program at the K4 level in two distinct roles: practitioners only and practitioners and program developers jointly. The study group included 66 teachers, 26 of whom had actively contributed to the development of the earlySTEM program. Teachers in both roles were supported by teacher guides, student books and workshops throughout the 8-month long academic year. Data was collected at the end of the academic year through an open-ended survey. The program developer teachers identified more student outcomes under more diverse categories while the practitioner teachers mainly concentrated on cognitive outcomes and limited their conceptualizations to the national curriculum. In addition, the program developer teachers valued their involvement in the program development process and expressed more diverse professional outcomes referring to different types of teacher knowledge

Mice deficient in the putative phospholipid flippase ATP11C exhibit altered erythrocyte shape, anemia, and reduced erythrocyte life span

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11Cshowed a lower rate ofPStranslocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserineexposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.This work was supported in part by National Health and Medical Research Council Grant GNT1061288. Supported by National Health and Medical Research Council Career Development Fellowship GNT1035858 and by the Ramaciotti Foundation

Visualizing the role of Cbl-b in control of islet-reactive CD4 T cells and susceptibility to Type 1 Diabetes

The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against Type 1 diabetes, but its in vivo role in the process of self-tolerance has not been examined at the level of potentially auto-aggressive CD4+ T cells. Here we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme antigen expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4+ T cells in pre-diabetic 3A9-TCR x insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency because it does not affect thymic negative selection of islet-reactive CD4+ cells nor the numbers of islet-specific CD4+ or CD4+ FOXP3+ T cells in the periphery, although it decreased differentiation of inducible Treg (iTreg) cells from TGF-b treated 3A9-TCR cells in vitro. When removed from Tregs and placed in culture, Cblb-deficient islet-reactive CD4+ cells reveal a capacity to proliferate to HEL antigen that is repressed in wild-type cells. This latent failure of T cell anergy is nevertheless controlled in vivo in pre-diabetic mice, so that islet-reactive CD4+ cells in spleen and pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb-deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wks of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance, and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity

IgD attenuates the IgM-induced anergy response in transitional and mature B cells

Self-tolerance by clonal anergy of B cells is marked by an increase in IgD and decrease in IgM antigen receptor surface expression, yet the function of IgD on anergic cells is obscure. Here we define the RNA landscape of the in vivo anergy response, comprising 220 induced sequences including a core set of 97. Failure to co-express IgD with IgM decreases overall expression of receptors for self-antigen, but paradoxically increases the core anergy response, exemplified by increased Sdc1 encoding the cell surface marker syndecan-1. IgD expressed on its own is nevertheless competent to induce calcium signalling and the core anergy mRNA response. Syndecan-1 induction correlates with reduction of surface IgM and is exaggerated without surface IgD in many transitional and mature B cells. These results show that IgD attenuates the response to self-antigen in anergic cells and promotes their accumulation. In this way, IgD minimizes tolerance-induced holes in the pre-immune antibody repertoire.This work was supported by NIH grant U19 AI100627 and NHMRC grants 585490, 1016953 and 1081858 to C.C.G., NHMRC CJ Martin Fellowship 595989 to J.H.R., an Endeavour Award from the Australian Government to Z.S. and the National Collaborative Research Infrastructure Scheme Australian Phenomics Facilit

IgD attenuates the IgM-induced anergy response in transitional and mature B cells

Self-tolerance by clonal anergy of B cells is marked by an increase in IgD and decrease in IgM antigen receptor surface expression, yet the function of IgD on anergic cells is obscure. Here we define the RNA landscape of the in vivo anergy response, comprising 220 induced sequences including a core set of 97. Failure to co-express IgD with IgM decreases overall expression of receptors for self-antigen, but paradoxically increases the core anergy response, exemplified by increased Sdc1 encoding the cell surface marker syndecan-1. IgD expressed on its own is nevertheless competent to induce calcium signalling and the core anergy mRNA response. Syndecan-1 induction correlates with reduction of surface IgM and is exaggerated without surface IgD in many transitional and mature B cells. These results show that IgD attenuates the response to self-antigen in anergic cells and promotes their accumulation. In this way, IgD minimizes tolerance-induced holes in the pre-immune antibody repertoire