Claude Saint-Ruf

Pleiotropic changes controlled by the pre-T-cell receptor.

The construction of various gene-deficient mice has facilitated the understanding of the role of various receptors and signaling pathways that control the generation of alphabeta lineage cells. A predominant role is occupied by the pre-TCR, which not only generates large numbers of alphabeta lineage cells but also controls TCRbeta allelic exclusion as well as commitment to the gammadelta lineage versus the alphabeta lineage.

Different initiation of pre-TCR and γδTCR signalling

Lineage choice is of great interest in developmental biology. In the immune system, the αβ and γδ lineages of T lymphocytes diverge during the course of the β-, γ- and δ-chain rearrangement of T-cell receptor (TCR) genes that takes place within the same precursor cell and which results in the formation of the γδTCR or pre-TCR proteins. The pre-TCR consists of the TCRβ chain covalently linked to the pre-TCRα protein, which is present in immature but not in mature T cells which instead express the TCRα chain. Animals deficient in pre-TCRα have few αβ lineage cells but an increased number of γδ T cells. These γδ T cells exhibit more extensive TCRβ rearrangement than γδ T cells from wild-type mice. These observations are consistent with the idea that different signals emanating from the γδTCR and pre-TCR instruct lineage commitment. Here we show, by using confocal microscopy and biochemistry to analyse the initiation of signalling, that the pre-TCR but not the γδTCR colocalizes with the p56lck Src kinase into glycolipid-enriched membrane domains (rafts) apparently without any need for ligation. This results in the phosphorylation of CD3ε and Zap-70 signal transducing molecules. The results indicate clear differences between pre-TCR and γδTCR signalling.

Crucial function of the pre‐T‐cell receptor (TCR) in TCRP selection, TCRβ allelic exclusion and αβ versus γδ lineage commitment

Summary: The analysis of T‐cell receptor (TCR) βselection, TCRβ allelic exclusion and TCRβ rearrangement in γδ T cells from normal and pre‐TCR‐deficient mice has shown that the pre‐TCR has a crucial role in T‐lyinpbocyte development:

On the Role of the Pre–T Cell Receptor in αβ versus γδ T Lineage Commitment

Abstract The role of the pre–T cell receptor (TCR) in lineage commitment to the γδ versus αβ lineage of T cells was addressed by analyzing TCRβ chain rearrangements in γδ T cells from wild-type and pre-TCR-deficient mice by single cell polymerase chain reaction. Results show that the pre-TCR selects against γδ T cells containing rearranged Vβ genes and that γδ T cell precursors but not γδ T cells express the pre-TCRα protein. Furthermore, pre-TCR-induced proliferation could not be detected in γδ T cells. We propose that the pre-TCR commits developing T cells to the αβ lineage by an instructive mechanism that has largely replaced an evolutionary more ancient stochastic mechanism of lineage commitment.

Genomic structure of the human pre-T cell receptor α chain and expression of two mRNA isoforms

The pre‐TCR, which is minimally composed of the TCRβ chain, the pre‐Tα chain, and the CD3 complex, regulates early T cell development. The pre‐Tα chain is a 33‐kDa type I transmembrane glycoprotein with an extracellular part similar to the constant domain of the immunoglobulin supergene family. We have sequenced (11 kb) the human pTα gene, which like the murine pTα gene consists of four exons: exon 1 encodes the 5 ′ untranslated region, the leader peptide and the first three amino acids of the mature protein, exon 2 the extracellular immunoglobulin (Ig)‐like domain, exon 3 a 15‐amino acid peptide including a cysteine required for heterodimerization with TCRβ, exon 4 the transmembrane region, the cytoplasmic tail and the 3 ′ untranslated sequence. The human pTα gene is located on chromosome 6p21.3, close to the HLA‐A locus. Reverse transcription‐PCR studies with human thymus and leukemic cells showed that alternative splicing produces a shorter pTα isoform, which lacks the Ig‐like domain but contains the transmembrane elements and the extracytoplasmic cystein and which could thus permit pairing with TCRβ chain and association with CD3 molecules. The conserved splice sites suggest a yet ill‐defined biological function of the short pTα protein.

Genomic structure and chromosomal location of the mouse pre-T-cell receptor alpha gene.

The mouse pre-T-cell receptor alpha (pTα) chain is a 33 000 Mr glycoprotein expressed on the surface of immature thymocytes as a disulfide-linked heterodimer with the T-cell receptor beta (TCRβ) chain, and in association with proteins of the CD3 complex. The cDNA for pTα, isolated previously, encodes a type I transmembrane protein that is a member of the immunoglobulin (Ig) superfamily. Here we report the complete nucleotide sequence, the exon/intron structure, and the chromosomal location of the pTa gene. The gene spans about 8.4 kilobases (kb) and consists of four exons. Exon 1 encodes the 5′ untranslated region, the leader peptide, and the first three amino acids of the mature protein. This exon is followed by a relatively long intron of 4.9 kb that contains many short interspersed repeats (SINEs) of the B1 and B2 family. The second exon encodes the extracellular Ig-like domain and exon 3 with just 45 base pairs the connecting peptide (CP), including the cysteine required for heterodimer formation. A similar exon/intron structure encoding corresponding parts of the mature polypeptide is found both in the Tcra and Tcrd constant region genes. The last exon encodes the transmembrane portion, the cytoplasmic tail, and about 540 nucleotides of 3′ untranslated sequence, including a B2 repetitive element. In situ hybridization maps the pTa gene to the D/E1 region of mouse chromosome 17.

Antibiotic Susceptibility Testing of the Gram-Negative Bacteria Based on Flow Cytometry

Rapidly treating infections with adequate antibiotics is of major importance. This requires a fast and accurate determination of the antibiotic susceptibility of bacterial pathogens. The most frequently used methods are slow because they are based on the measurement of growth inhibition. Faster methods, such as PCR-based detection of determinants of antibiotic resistance, do not always provide relevant information on susceptibility, particularly that which is not genetically based. Consequently, new methods, such as the detection of changes in bacterial physiology caused by antibiotics using flow cytometry and fluorescent viability markers, are being explored. In this study, we assessed whether Alexa Fluor® 633 Hydrazide (AFH), which targets carbonyl groups, can be used for antibiotic susceptibility testing. Carbonylation of cellular macromolecules, which increases in antibiotic-treated cells, is a particularly appropriate to assess for this purpose because it is irreversible. We tested the susceptibility of clinical isolates of Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, to antibiotics from the three classes: β-lactams, aminoglycosides, and fluoroquinolones. In addition to AFH, we used TO-PRO®-3, which enters cells with damaged membranes and binds to DNA, and DiBAC4 (3), which enters cells with depolarized membranes. We also monitored antibiotic-induced morphological alterations of bacterial cells by analyzing light scattering signals. Although all tested dyes and light scattering signals allowed for the detection of antibiotic-sensitive cells, AFH proved to be the most suitable for the fast and reliable detection of antibiotic susceptibility.

Strong increase in the autofluorescence of cells signals struggle for survival

Prokaryotic and eukaryotic cells exhibit an intrinsic natural fluorescence due to the presence of fluorescent cellular structural components and metabolites. Therefore, cellular autofluorescence (AF) is expected to vary with the metabolic states of cells. We examined how exposure to the different stressors changes the AF of Escherichia coli cells. We observed that bactericidal treatments increased green cellular AF, and that de novo protein synthesis was required for the observed AF increase. Excitation and emission spectra and increased expression of the genes from the flavin biosynthesis pathway, strongly suggested that flavins are major contributors to the increased AF. An increased expression of genes encoding diverse flavoproteins which are involved in energy production and ROS detoxification, indicates a cellular strategy to cope with severe stresses. An observed increase in AF under stress is an evolutionary conserved phenomenon as it occurs not only in cells from different bacterial species, but also in yeast and human cells.

Depletion of ZBTB38 potentiates the effects of DNA demethylating agents in cancer cells via CDKN1C mRNA up-regulation

DNA methyltransferase inhibitor (DNMTi) treatments have been used for patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and have shown promising beneficial effects in some other types of cancers. Here, we demonstrate that the transcriptional repressor ZBTB38 is a critical regulator of the cellular response to DNMTi. Treatments with 5-azacytidine, or its derivatives decitabine and zebularine, lead to down-regulation of ZBTB38 protein expression in cancer cells, in parallel with cellular damage. The depletion of ZBTB38 by RNA interference enhances the toxicity of DNMTi in cell lines from leukemia and from various solid tumor types. Further we observed that inactivation of ZBTB38 causes the up-regulation of CDKN1C mRNA, a previously described indirect target of DNMTi. We show that CDKN1C is a key actor of DNMTi toxicity in cells lacking ZBTB38. Finally, in patients with MDS a high level of CDKN1C mRNA expression before treatment correlates with a better clinical response to a drug regimen combining 5-azacytidine and histone deacetylase inhibitors. Collectively, our results suggest that the ZBTB38 protein is a target of DNMTi and that its depletion potentiates the toxicity of DNMT inhibitors in cancer cells, providing new opportunities to enhance the response to DNMT inhibitor therapies in patients with MDS and other cancers.