Expression of TCR-Vß 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-Vß8.2 in absence of anti-Cß antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vß8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vß8.2(+) Cß(-) 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(-) Vß8.2(+) Cß(-) 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(-) Vß8.2(+) Cß(-) BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.

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-V beta 8.2 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-V beta 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 germline TCR-Vbeta transcripts in extrathymic lymphoid sites.

Germline TCR-Vbeta transcription is commonly considered an event coupled with rearrangement of TCR genes in T cells. The extent of germline Vbeta transcription is studied here in a range of cell types and in several mouse strains. A sensitive semi-quantitative RT-PCR method was developed to specifically detect germline and not rearranged transcripts. Germline transcription of a range of different Vbeta genes was detected along with rearranged transcripts in bone marrow, thymus, mesenteric lymph node and spleen. Some transcripts were also detected in low level in non-lymphoid tissues including heart, liver and brain. Expression was also studied in the C57BL/6J-beta2microglobulin-/- (C57BL/6J-beta2M-/-) mouse model that lacks NK1.1 T cells and predominantly utilises Vbeta8.2 in the formation of a TCR. beta2M-/- mice, which lack both CD1-dependent NK1.1 T cells and CD8+ T cells, showed germline TCR-Vbeta8 transcription in most tissues indicating that germline transcription is not specifically related to CD1-dependent NK1.1 T cells. In many tissues, multiple transcripts were amplified representing both spliced and unspliced forms of germline Vbeta. For most Vbeta genes, the expression of spliced and unspliced forms was equivalent. Given an abundance of unspliced transcripts, the presence of alternative ORFs encoding a novel protein was investigated within the TCR-Vbeta genes. Sequence analysis of ORFs showed only genes with a high level of similarity to TCR-beta. All data reflect the prevalence of germline transcripts in vivo and raise questions about their functional role.

Cell surface expression of a peptide encoded by the unrearranged TCR-Vbeta8.2 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 Vbeta genes directs gene usage during the rearrangement event. Germline transcription of the TCR-Vbeta8.2 gene has been detected in a precursor T-cell line, C1-V13D, which shows no rearrangement at any of the TCR gene loci. This cell line also shows weak binding of specific anti-Vbeta8.2 antibody to the cell surface, consistent with expression of a truncated TCRbeta chain. RT-PCR has been used to confirm expression of spliced germline transcripts of TCR-Vbeta8.2 in C1-V13D initiated from both leader (L)5.1 and L8.2. Transcripts initiated from L8.2 were also detectable in unspliced form. In order to test expression and subcellular localisation of any encoded peptides, amplified germline transcripts in both spliced and unspliced form were cloned into the pEGFP-N1 fusion vector for stable transfection and overexpression in C1-V13D. Cell surface expression of a fusion protein between EGFP and a Vbeta peptide has been confirmed in C1-V13D but not in control COS-7 cells. Results presented here raise the possibility of a new pre-TCR structure specific to early lymphoid cells and based on TCR-Vbeta8.2 gene expression.

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.

Germline transcription of multiple TCR-Vbeta 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-Vbeta genes might be expected in early T cells which have not yet undergone rearrangement. Germline transcription of TCR-Vbeta 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-Vbeta8.2 and TCR-Vbeta2.1 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 Vbeta2.1, Vbeta5.1, Vbeta5.2, Vbeta6.1, Vbeta7.1, Vbeta8.1, Vbeta8.2, Vbeta8.3 and Vbeta13.1. However, D10.G4.1 cells expressed a subset of transcripts with apparently lower levels of expression, including Vbeta2.1, Vbeta5.1, Vbeta5.2, Vbeta6.1, Vbeta8.2 and Vbeta8.3. 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.

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.