Expression of TCR-Vβ peptides by murine bone marrow cells does not identify T-cell progenitors

Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vβ8.2 in absence of anti-Cβ antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vβ8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vβ8.2(+) Cβ(-) are found in several lymphoid sites, and among the lineage-negative (Lin(-) ) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin(-) Vβ8.2(+) Cβ(-) BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin(-) Vβ8.2(+) Cβ(-) BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.This study was supported by grants to H.C.O. from the National Health and Medical Research Council of Australia. J.A. was supported by a fellowship from the Australian Academy of Science

The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum

BACKGROUND: The uptake of nutrients, expulsion of metabolic wastes and maintenance of ion homeostasis by the intraerythrocytic malaria parasite is mediated by membrane transport proteins. Proteins of this type are also implicated in the phenomenon of antimalarial drug resistance. However, the initial annotation of the genome of the human malaria parasite Plasmodium falciparum identified only a limited number of transporters, and no channels. In this study we have used a combination of bioinformatic approaches to identify and attribute putative functions to transporters and channels encoded by the malaria parasite, as well as comparing expression patterns for a subset of these. RESULTS: A computer program that searches a genome database on the basis of the hydropathy plots of the corresponding proteins was used to identify more than 100 transport proteins encoded by P. falciparum. These include all the transporters previously annotated as such, as well as a similar number of candidate transport proteins that had escaped detection. Detailed sequence analysis enabled the assignment of putative substrate specificities and/or transport mechanisms to all those putative transport proteins previously without. The newly-identified transport proteins include candidate transporters for a range of organic and inorganic nutrients (including sugars, amino acids, nucleosides and vitamins), and several putative ion channels. The stage-dependent expression of RNAs for 34 candidate transport proteins of particular interest are compared. CONCLUSION: The malaria parasite possesses substantially more membrane transport proteins than was originally thought, and the analyses presented here provide a range of novel insights into the physiology of this important human pathogen

Expression of T-cell receptor genes during early T-cell development

Lymphoid cell development is an ordered process that begins in the embryo in specific sites and progresses through multiple differentiative steps to production of T- and B-cells. Lymphoid cell production is marked by the rearrangement process, which gives rise to mature cells expressing antigen-specific T-cell receptors (TCR) and immunoglobulins (Ig). While most transcripts arising from TCR or Ig loci reflect fully rearranged genes, germline transcripts have been identified, but these have always been thought to have no specific purpose. Germline transcription from either unrearranged TCR or unrearranged Ig loci was commonly associated with an open chromatin configuration during VDJ recombination. Since only early T and B cells undergo rearrangement, the association of germline transcription with the rearrangement process has served as an appropriate explanation for expression of these transcripts in early T- and B-cell progenitors. However, germline TCR-Vβ8.2 transcripts have now been identified in cells from RAG-/- mice, in the absence of the VDJ rearrangement event and recombinase activity. Recent data now suggest that germline TCR-Vβ transcription is a developmentally regulated lymphoid cell phenomenon. Germline transcripts could also encode a protein that plays a functional role during lymphoid cell development. In the least, germline transcripts serve as markers of early lymphoid progenitors

Detection of spliced and unspliced forms of germlineTCR-VB transcripts in extrathymic lymphoid sites

Germline TCR-Vβ transcription is commonly considered an event coupled with rearrangement of TCR genes in T cells. The extent of germline Vβ transcription is studied here in a range of cell types and in several mouse strains. A sensitive semi-quantitati

Germline transcription of multiple TCR-V� genes in cloned T-cell lines

The functional significance of germline transcription of T cell receptor (TCR) beta chain variable (V) region genes is under investigation. The accepted model is that transcriptional activation of germline TCR genes is associated with the rearrangement process during T-cell development. By this model, germline expression of a subset of TCR- Vβ genes might be expected in early T cells which have not yet undergone rearrangement. Germline transcription of TCR- Vβ genes was analysed using the reverse transcriptase (RT)-PCR in a clonal T-cell precursor line C1-V13D, a clonal pre-B cell line RAW112 and a mature T helper cell line D10.G4.1. Evidence is presented for germline transcription of TCR-Vβ8.2 and TCR-Vβ2.1 genes in all three cell lines, although expression in RAW112 was very weak. C1-V13D cells expressed very high levels of the whole range of transcripts including Vβ2.1, Vβ5.1, Vβ5.2, Vβ6.1, Vβ7.1, Vβ8.1, Vβ8.2, Vβ8.3 and Vβ13.1. However, D10.G4.1 cells expressed a subset of transcripts with apparently lower levels of expression, including Vβ2.1, Vβ5.1, Vβ5.2, Vβ6.1, Vβ8.2 and Vβ8.3. These results raise questions about the significance and possible function of germline transcripts and/or their encoded products in early lymphoid cells and in T cells at different stages of development

Cell surface expression of a peptide encoded by the unrearranged TCR-Vß8.2 gene

Germline transcription of T-cell receptor (TCR) genes has been described in early lymphoid cells. The most common explanation for this phenomenon is that transcription of unrearranged Vβ genes directs gene usage during the rearrangement event. Germline

Gene signature of Stromal Cells which support Dendritic Cell Development

Spleen stromal cells are critical determinants of dendritic cell (DC) development in spleen. The spleen stromal line, namely STX3, supports DC differentiation in vitro from overlaid bone marrow cells while the lymph node stromal line, namely 2RL22, does not. Here we have characterised the hematopoietic support capacity of each stroma, and analysed lineage origin of the stromal cell lines by gene profiling using microarrays. Stromal co-culture experiments were performed using bone marrow cells as a source of hematopoietic progenitors. A characteristic immature myeloid-like CD11c+CD11b +CD86+MHC-II-/loB220-CD8α - DC is produced after 14 days in STX3 cocultures, while 2RL22 cocultures produce only monocyte/macrophage-like cells. No other hematopoietic cell type is produced. The STX3 and 2RL22 stroma were compared by transcriptome analysis utilising Affymetrix Murine U74Av2 genechips to identify gene expression related to differential hematopoietic support function. Data mining was used to determine cell surface marker expression reflecting endothelial cells and fibroblasts, as well as adhesion molecules contributing to the microenvironment. STX3 shows gene expression reflective of early endothelial cells, while 2RL22 expresses markers specific to fibroblasts. The expression of genes like Flt1, CD34, Mcam, and Eng distinguishes STX3 as an early immature endothelial cell lacking markers of angioblasts or hemangioblasts like Tal1/SCL, Tie1, Tie2, Kdr or Prom1/AC133. The absence of expression of genes like Vwf and Cd31 distinguishes STX3 from fully differentiated vascular endothelial cells. In contrast, the 2RL22 lymph node stroma specifically expresses genes related to fibroblastic-like cells like osteoblasts with expression of Vdr (Vitamin D receptor), and epithelial cells with expression of Krt13 (keratins). Gene expression data identifies STX3 as splenic endothelial cells, independently able to support the outgrowth of immature, myeloid DC-like cells from progenitors present in bone marrow, while 2RL22 lymph node fibroblastic cells provide support for development of monocytes/macrophages

Dendritic Cell Development in Long-Term Spleen Stromal Cultures

The cellular microenvironments in which dendritic cells (DCs) develop are not known. DCs are commonly expanded from CD34+ bone marrow precursors or blood monocytes using a cocktail of growth factors including GM-CSF. However, cytokine-supported cultures are not suitable for studying the intermediate stages of DC development, since progenitors are quickly driven to become mature DCs that undergo limited proliferation and survive for only a short period of time. This lab has developed a long-term culture (LTC) system from spleen which readily generates a high yield of DCs. Hematopoietic cells develop under more normal physiological conditions than in cultures supplemented with cytokines. A spleen stromal cell monolayer supports stem cell maintenance, renewal, and the specific differentiation of only DCs and no other hematopoietic cells. Cultures maintain continuous production of a small population of small-sized progenitors and a large population of fully developed DCs. Cell-cell interaction between stromal cells and progenitor cells is critical for DC differentiation. The progenitors maintained in LTC appear to be quite distinct from bone marrow-derived DC progenitors that respond to GM-CSF. The majority of cells produced in LTC are large-sized cells with a phenotype reflecting myeloid-like DC precursors or immature DCs. These cells are highly endocytotic and weakly immunostimulatory for T cells. This model system predicts in situ production of DCs in spleen from endogenous progenitors, as well as a central role for spleen in DC hematopoiesis