Global gene expression analysis of human erythroid progenitors

This article is available open access through the publisher’s website. Copyright @ 2011 American Society of Hematology. This article has an erratum: http://bloodjournal.hematologylibrary.org/content/118/26/6993.3.Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html.National Health Service Blood and Transplant, National Institute for Health Research Biomedical Research Center Program, and National Institute for Health Research

Visualization and Identification of IL-7 Producing Cells in Reporter Mice

Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging

Visualization and Identification of IL-7 Producing Cells in Reporter Mice

Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging

Transcriptomic analysis supports similar functional roles for the two thymuses of the tammar wallaby

Background: The thymus plays a critical role in the development and maturation of T-cells. Humans have a single thoracic thymus and presence of a second thymus is considered an anomaly. However, many vertebrates have multiple thymuses. The tammar wallaby has two thymuses: a thoracic thymus (typically found in all mammals) and a dominant cervical thymus. Researchers have known about the presence of the two wallaby thymuses since the 1800s, but no genome-wide research has been carried out into possible functional differences between the two thymic tissues. Here, we used pyrosequencing to compare the transcriptomes of a cervical and thoracic thymus from a single 178 day old tammar wallaby.Results: We show that both the tammar thoracic and the cervical thymuses displayed gene expression profiles consistent with roles in T-cell development. Both thymuses expressed genes that mediate distinct phases of T-cells differentiation, including the initial commitment of blood stem cells to the T-lineage, the generation of T-cell receptor diversity and development of thymic epithelial cells. Crucial immune genes, such as chemokines were also present. Comparable patterns of expression of non-coding RNAs were seen. 67 genes differentially expressed between the two thymuses were detected, and the possible significance of these results are discussed.Conclusion: This is the first study comparing the transcriptomes of two thymuses from a single individual. Our finding supports that both thymuses are functionally equivalent and drive T-cell development. These results are an important first step in the understanding of the genetic processes that govern marsupial immunity, and also allow us to begin to trace the evolution of the mammalian immune system

Natural killer cell immunity after transplantation

Transplantation immunology has traditionally focused on adaptive, i.e., T- and B-cell reactions. More recently, natural killer (NK) cells were also recognised as playing an important role after transplantation of solid organs and haematopoietic stem cells. NK cells recognise "cell stress" induced by viral replication and tumour transformation via activating receptors, and are negatively regulated by the interaction between inhibitory molecules and autologous human leukocyte antigens (HLA). The most important inhibitory molecules belong to the family of killer cell immunoglobulin-like receptors (KIR). Differences in the inhibitory KIR/HLA interaction between stem cell donor and patient may lead to beneficial NK cell alloreactivity, resulting in specific graft-versus-tumour reactions, which occur in the absence of graft-versus-host disease. The immaturity of NK cells produced by the stem cell graft early after transplantation has led to different approaches of adoptive transfer of NK cells to further increase tumour control. The function and role of activating KIR receptors is less clear. Recent data have suggested, that activating KIR may also contribute to anti-tumour immunity after stem cell transplantation, as patients transplanted from donors carrying high numbers of activating KIR receptor genes show reduced relapse rates. In particular, protection from post-transplant disease relapse was demonstrated in transplants carried out from donors carrying the activating KIR2DS1 receptor, if the recipients also expressed the KIR2DS1 ligand HLA-C2. In conclusion, NK cells have been firmly established in the last two decades as relevant players in transplant immunology, which can critically determine the outcome of haematopoietic stem cell grafts

A comprehensive analysis of the binding of anti-KIR antibodies to activating KIRs

Analysis of killer cell immunoglobulin-like receptor (KIR) expression has been notoriously difficult because of the cross-reactivity of available antibodies, in particular between activating and inhibitory isoforms. We undertook a comprehensive study of available anti-KIR antibodies binding to activating KIRs (a-KIRs). Using cell lines stably transfected with a-KIRs (KIR2DS1-S5 and KIR3DS1), we confirmed documented binding specificities. In addition, we show that clones HPMA4 and 143211-previously assumed to be specific for KIR2DS1/L1 and KIR2DL1, respectively-bind KIR2DS5 and KIR2DS3 (HPMA4), and KIR2DS5 (143211). Other antibodies with previously undocumented binding were JJC11.6 (recognizing KIR2DS3) and 5.133 (recognizing all a-KIRs except KIR2DS1 and KIR2DS3). The novel KIR2DS5 reactivities were confirmed by blocking with soluble KIR-Fc fusion proteins, and by reverse transcriptase-PCR analysis of sorted primary natural killer cells. In conclusion, we show formerly undocumented binding properties of anti-KIR antibodies. These cross-reactivities should be taken into account when analyzing KIR expression.Genes and Immunity advance online publication, 31 October 2013; doi:10.1038/gene.2013.58