Cécile Tonnelle

Age-related changes in human hematopoietic stem/progenitor cells

Adult stem cells are critical for maintaining cellular homeostasis throughout life, yet the effects of age on their regenerative capacity are poorly understood. All lymphoid and myeloid blood cell lineages are continuously generated from hematopoietic stem cells present in human bone marrow. With age, significant changes in the function and composition of mature blood cells are observed. In this study, we report that age‐related changes also occur in the human hematopoietic stem cell compartment. We find that the proportion of multipotent CD34+CD38− cells increases in the bone marrow of elderly (>70 years) individuals. CD34+CD38+CD90−CD45RA+/−CD10− and CD34+CD33+ myeloid progenitors persist at the same level in the bone marrow, while the frequency of early CD34+CD38+CD90−CD45RA+CD10+ and committed CD34+CD19+ B‐lymphoid progenitors decreases with age. In contrast to mice models of aging, transplantation experiments with immunodeficient NOD/SCID/IL‐2Rγ null (NSG) mice showed that the frequency of NSG repopulating cells does not change significantly with age, and there is a decrease in myeloid lineage reconstitution. An age‐related decrease in the capacity of CD34+ cells to generate myeloid cells was also seen in colony‐forming assays in vitro. Thus, with increasing age, human hematopoietic stem/progenitor cells undergo quantitative changes as well as functional modifications.

Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.

We report in this paper the first analysis of the expression pattern of Ig VH and VK families in human adult normal peripheral B lymphocytes, by in situ hybridization using specific VH1 to VH6 and VK1 to VK4 probes, which cover the known human V gene families reported to date. The major families were VH3 and VK1, with the respective gradient VH3 greater than VH4 greater than VH1 greater than VH5 greater than VH6 greater than VH2, and VK1 greater than VK3 greater than VK4 greater than VK2. Using a large sampling of EBV clones, we found that the pattern of VH and VK family usage was similar. The expression level correlated fairly with the estimated gene number for the VH, but diverged noticeably for the K chains. Taken together with the fact that the level of light chain expression (K + lambda) was about two-fold that of heavy chains, these results suggest that the VH and the VK repertoires are not regulated by a similar selective process.

Fetal versus Adult PreB or B Cells: The Human VH Repertoirea

At the preB stage, when only the IGH locus has rearranged, mu chains become expressed in association with the psi L chains, lambda-like and VpreB, thus forming the preB receptor. By the use of a monoclonal anti VpreB antibody, preB cells were isolated from two adult bone marrow samples, and the VH repertoire was analyzed and compared to fetal, XLA (X-linked agammaglobulinemia), and adult B repertoires. Most VH genes identified were also expressed in fetal liver, XLA bone marrow, and adult PBLs, with similar predominant usage of certain germline genes. Multiple D/D fusions, limited N diversity, and preferential use of JH4 with a low level of DQ52 usage were also identified. Few mutations could be observed, not specifically localized in CDR regions, that could be interpreted as not positively selected. Conversely, a shorter length of CDR3 appeared to be the hallmark of the preB step. Thus, the association of psi L chains with mu does not bring about a bias in the VH gene usage, but a first selection on the CDR3 region could be the result of recognition by given autoantigens or ligands different for preB cells and B cells.

Stage specific over-expression of the dominant negative Ikaros 6 reveals distinct role of Ikaros throughout human B-cell differentiation

Ikaros is a transcription factor that acts both as an activator and as an inhibitor of gene expression in several hematopoietic lineages. Ikaros functions in hematopoiesis have mostly been studied in mice, and are notably crucial for lymphopoiesis. Deregulation of Ikaros expression was evidenced in several leukemia subtypes, including pre-B-ALL. Here, we studied the role of Ikaros in human B lymphoid differentiation through xeno-transplantation of genetically modified cord blood (CB) human hematopoietic progenitor cells (HPC) in NOD/SCID mice. We used lentiviral vectors to force expression of Ikaros 6 (Ik6), a known dominant negative (DN) protein that interferes with normal Ikaros and structurally related proteins in HPC and their progeny. Two types of vectors were used: a vector containing the EF1alpha promoter which produces strong gene expression in all hematopoietic lineages, and a recently validated B-specific vector containing an enhanced CD19 derived promoter that strongly favors expression in the B-cell lineage. Ik6 transduction of CB CD34(+) cells with these vectors produced distinct consequences in the B-cell differentiation profiles of xenografted human cells. While the ubiquitous vector favored a specific block at the early pro-B/pre-B stage of differentiation, with an increase in Lambda Like transcript expression in the bone marrow (BM), B-specific Ik6 expression provoked a global decrease in the CD19(+) cell population in both BM and spleen, associated with a decrease in IgM+ immature B-cells in the spleen. We conclude that Ikaros proteins are active throughout human B-cell differentiation, before and after CD19 appearance.

Potential Application of Gene Therapy to X-Linked Agammaglobulinemia

X-linked agammaglobulinemia (XLA), or Brutons disease, is the most common human primary humoral immunodeficiency. XLA is caused by mutations of the Brutons tyrosine kinase (BTK), a key regulator of B-cell physiology. Since the mid 80s, substitutive therapy by intravenous gammaglobulin infusions has significantly improved XLA patient survival and quality of life. Nevertheless, some frequent affections persist despite treatment, and lead to handicapping and further to morbid clinical complications for XLA individuals. Development of gene therapy by transfer of the BTK gene into hematopoietic progenitors could represent an alternative strategy for the treatment of Brutons disease, with the advantage of a definitive cure for XLA patients. Gene therapy of XLA could be considered as a paradigm for future expansion of gene therapy approaches for many other diseases, since future utilization may be strictly dependent on a marked improvement of risk-benefit ratio compared to pre-existing treatments.

NH2-terminal amino-acid sequences of poly (Glu60, Ala30, Tyr10) (GAT) and poly (Glu60, Ala40) (GA) monoclonal antibody heavy chains☆

Abstract The heavy-chain NH 2 -terminal sequences of five IgM, kappa monoclonal anti-GAT§ antibodies, and five IgG, kappa anti-GA antibodies (all derived from immune DBA/2 mice), have been determined. These monoclonal hybridoma antibodies were previously characterized with respect to their idiotypic properties. Extensive identity, covering possibly up to residue 33, was observed between the five IgM, kappa anti-GAT antibodies, whether they expressed the cross-reactive idiotypic specificities (CGAT) or not. One anti-GAT γ 1 -chain derived from (B6 × D2)F1 mice was blocked. Among the five CGAT-negative, monoclonal anti-GA antibodies from DBA/2 mice, strong homologies were observed between the four heavy-chain amino-acid sequences of the antibodies expressing the cross-reactive GA-1 idiotypic specificities. These NH 2 -terminal V H sequences differed from each other and from those of the anti-GAT monoclonal heavy chains from the DBA/2 strain by only one amino acid, on average. Conversely, of the three GA-1-negative anti-GA antibodies for which sequencing was attempted, two were blocked, and one differed sharply from the others.

Ikaros gene expression and leukemia.

The Ikaros (Ik) protein, or LyF1, was initially described as a protein binding to regulatory sequences of a number of genes expressed in murine lymphoid cells. Ikaros is a critical regulator of normal hematopoietic stem cell differentiation, as evidenced by dramatic defects in the lymphoid compartments, in homozygous animals with gene inactivation. Because differential splicing produces multiple isoforms with potentially different functions, Ikaros provides a unique model to study how post-transcriptional mechanisms may be involved in neoplastic processes. Indeed, several groups including ours have underlined evidences that expression of different Ikaros isoforms vary among different types of leukemias. The predominance of short isoforms in certain subsets is intriguing. Here, additional observations reinforced the hypothesis that Ikaros expression may be deregulated in human leukemias. Whether this is a cause or a consequence of the leukemic process remains speculative. Other human diseases however, provide examples of abnormal post-transcriptional regulations that have been further characterized.

IGMκ/λ EBV human B cell clone: An early step of differentiation of fetal B cells or A distinct B lineage?☆

Abstract In agreement with the clonal theory, one B lymphocyte synthetizes one antibody due to allelic and isotypic exclusion. We analyzed an EBV B-cell clone, E29.1, derived from an 11 week-old embryo, and secreting both IgMκ and IgMλ. Structural analysis of produced IgM, indicated that λ-containing pentamers could be considered hybrid molecules, expressing both the κ and the λ chains, with a κ/λ ratio between 5 and 10. It was also found that 60% of the λ chains were secreted in free form, presumably as a result of a better affinity of μ chains for κ chains. The sequence of the three transcripts had an entirely ORF (Open Reading Frame), and were very close to germline sequences, with, however, an additional codon between Vκ and Jκ gene which has never been described in adult myeloma protein or cDNA human sequence. This observation is suggestive of N diversity taking place in κ chains. The possible role of Kde (κ deleting element) recombination onto κ/λ locus activation was analyzed on a collection of 23 λ clones. The status of rearrangement of κ genes indicated that 35% of these clones had retained, at least, one κ allele without the Kde recombination, four λ clones had one κ allele in germline configuration. Different hypotheses of maturation from pre-B cell to B cell with activation of light chain genes are discussed.

Structural correlates to the rabbit immunoglobulin heavy chain a100 allotype

Three a100/a100 homozygous rabbits immunized with Micrococcus lysodeikticus produced large amounts of anti-polysaccharide antibodies of restricted heterogeneity. These antibodies were purified by either immunoabsorption or ion exchange chromatography. The almost complete sequence of one heavy chain spanning residues 1-123, with the exception of 10 residues (66-67 and 79-86), was determined. Partial sequence data were also obtained for the two other heavy chains. The identity of these three sequences in the first framework region unraveled a prototype sequence of the a100 allotype that differs from homologous sequence of VH regions that determine other allotypic specificities. The gradient of sequence conservation was found to be a100 greater than a3 greater than a1 greater than a- greater than a2. Homologies in sequence paralleled previously described serological cross-reactions observed between a100, a3 and a1 specificities. This remarkable conservation of framework residues suggests that the VH regions of the rabbit immunoglobulins represent a paucigene system, in which each basic allotypic specificity might be encoded a discrete subgroup of genes.

Expression and recombination of the EGFP and EYFP genes in lentiviral vectors carrying two heterologous promoters.

BACKGROUND Expressing two genes in the progeny of stem and progenitor cells that are transduced with a unique viral vector is desirable in certain situations. We tested the ability of two lentiviral vectors to transduce human cells of hematopoietic origin and concomitantly express two reporter genes, either EGFP (enhanced green fluorescent protein) and DsRed2, or EGFP and EYFP (enhanced yellow fluorescent protein), from two internal promoters. METHODS The vectors were generated from the pTRIP deltaU3 EF1alpha EGFP lentiviral vector. Following transduction of hematopoietic and non-hematopoietic cell lines, we performed FACS, PCR and Southern blot analyzes to quantify transduction, integration efficiencies and size of integrated lentiviral vectors, respectively. RESULTS The detection of DsRed2 fluorescence appeared unexpectedly low in human cells of hematopoietic origin. Alternatively, a modification in the flow cytometry assay allowed us to distinguish between the two overlapping fluorescence signals emitted by EGFP and EYFP, when transduced cells were excited with a 488-nm laser beam. However, the low frequency of double-positive EGFP+ EYFP+ cells, and the existence of single-positive, mostly EGFP- EYFP+, cells, prompted us to search for recombinations in the vector sequence. Southern blotting of DNA obtained from transduced cells indeed demonstrated that recombination had occurred between the two closely related EGFP and EYFP sequences. DISCUSSION These observations suggest that recombination occurred within the EGFP and EYFP genes, which differ by only four amino acids. We conclude that the insertion of two highly homologous sequences into a lentiviral backbone can favor recombination.