A human postnatal lymphoid progenitor capable of circulating and seeding the thymus

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34+CD10+ progenitor population and which is distinct from B cell–committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin−CD34+CD10+CD24− progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor α, and CD3ε. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus

DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer

Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160 232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy—in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution

Real-Time Definition of Non-Randomness in the Distribution of Genomic Events

Features such as mutations or structural characteristics can be non-randomly or non-uniformly distributed within a genome. So far, computer simulations were required for statistical inferences on the distribution of sequence motifs. Here, we show that these analyses are possible using an analytical, mathematical approach. For the assessment of non-randomness, our calculations only require information including genome size, number of (sampled) sequence motifs and distance parameters. We have developed computer programs evaluating our analytical formulas for the real-time determination of expected values and p-values. This approach permits a flexible cluster definition that can be applied to most effectively identify non-random or non-uniform sequence motif distribution. As an example, we show the effectivity and reliability of our mathematical approach in clinical retroviral vector integration site distribution

Mutating RBF Can Enhance Its Pro-Apoptotic Activity and Uncovers a New Role in Tissue Homeostasis

International audienceThe tumor suppressor retinoblastoma protein (pRb) is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD) is found in the Drosophila homologue of pRb (RBF) at a position similar to the site generating the p76Rb form in mammals. Thus, we generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBF D253A , conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. We show that this overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg) ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo

Polygenic mutations in the cytotoxicity pathway increase susceptibility to develop HLH immunopathology in mice

International audienceHemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disease. Inherited forms of HLH are caused by biallelic mutations in several effectors of granule-dependent lymphocyte-mediated cytotoxicity. A small proportion of patients with a so-called "secondary" form of HLH, which develops in the aftermath of infection, autoimmunity, or cancer, carry a monoallelic mutation in one or more HLH-associated genes. Although this observation suggests that HLH may have a polygenic mode of inheritance, the latter is very difficult to prove in humans. In order to determine whether the accumulation of partial genetic defects in lymphocyte-mediated cytotoxicity can contribute to the development of HLH, we generated mice that were doubly or triply heterozygous for mutations in HLH-associated genes, those coding for perforin, Rab27a, and syntaxin-11. We found that the accumulation of monoallelic mutations did indeed increase the risk of developing HLH immunopathology after lymphocytic choriomeningitis virus infection. In mechanistic terms, the accumulation of heterozygous mutations in the two degranulation genes Rab27a and syntaxin-11, impaired the dynamics and secretion of cytotoxic granules at the immune synapse of T lymphocytes. In addition, the accumulation of heterozygous mutations within the three genes impaired natural killer lymphocyte cytotoxicity in vivo. The genetic defects can be ranked in terms of the severity of the resulting HLH manifestations. Our results form the basis of a polygenic model of the occurrence of secondary HLH

RBF- and RBF^D253A-induced apoptosis as well as RBF^D253A-induced overgrowth depend on the JNK pathway activity.

<p>(A–C) Anti-◯P-JNK staining in young third instar larvae wing imaginal discs. (A) No staining is observed in <i>ptc-Gal4/+</i> control. (B, C) <i>UAS-RBF/ptc-Gal4</i> and <i>UAS-RBF^D253A/X; ptc-Gal4/+</i> discs present anti-◯P-JNK staining at the dorso-ventral boundary. All discs are shown with posterior to the top. (D) Notch phenotypes in adult wings of <i>vg-Gal4/+</i>; <i>UAS-RBF</i> and <i>vg-Gal4/+</i>; <i>UAS-RBF/UAS-bsk-RNAi</i> flies are grouped into four categories (wild type, weak, intermediate and strong) according to the number and size of notches observed on the wing margin (asterisks). <i>Bsk-RNAi</i> partially suppresses <i>RBF</i>-induced notch phenotypes (Wilcoxon test, α<10⁻¹⁵, n = 540). (E) The frequency of RBF^D253A-induced ectopic tissue growth is strongly decreased in <i>UAS-bsk-RNAi</i> co-expressing flies (Chi² test, α = 8.8 10⁻¹⁵). (F) TUNEL-labeling of apoptotic cells in wing imaginal discs; specific staining of apoptotic cells corresponds to bright patches in wing discs <i>of vg-Gal4/+</i>; <i>UAS-bsk-RNAi/+</i> (left panel), <i>vg-Gal4/+</i>; <i>UAS-RBF^D253A/+</i> (center panel), <i>vg-Gal4/+</i>; <i>UAS-RBF^D253A/UAS-bsk-RNAi</i> (right panel) <i>larvae</i>. RNAi-mediated knockdown of <i>bsk</i> strongly decreased RBF^D253A-induced apoptosis. All discs are shown with posterior to the top.</p

RBF^D253A is pro-apoptotic in the ZNC and induces more apoptosis than RBF in third instar larvae wing imaginal discs.

<p>(A, E) <i>C96-Gal4</i> and <i>vg-Gal4</i> expression patterns are visualized by <i>UAS-mtGFP</i> expression in third instar larvae wing imaginal discs. (B-D, F-H) Apoptotic cells are labeled by TUNEL in wing imaginal discs; specific staining of apoptotic cells corresponds to bright white patches. (B, F) <i>C96-Gal4/+</i> and <i>vg-Gal4/+</i> control discs have few apoptotic cells. (C) <i>C96-Gal4/UAS-RBF</i> wing discs are similar to control. (D) Some apoptotic cells are observed within the <i>C96-Gal4</i> expression domain in <i>UAS-RBF^D253A/X</i>; <i>C96-Gal4/+</i> discs (white arrow). (G, H) Apoptotic cells are observed within the <i>vg-Gal4</i> expression domain in <i>vg-Gal4/+</i>; <i>UAS-RBF/+</i> and <i>UAS-RBF^D253A/X</i>; <i>vg-Gal4/+</i> wing discs (white arrows). (G) The white arrowhead indicates a zone at the center of the pouch where cells are not TUNEL-labeled in <i>vg-Gal4/+</i>; <i>UAS-RBF/+</i> wing discs. All discs are shown with posterior to the top.</p

RBF^D253A-induced overgrowth does not depend on Wg.

<p>(A, B) Anti-Wg staining in wing imaginal discs of control (A) or <i>en-Gal4, UAS-wg-RNAi</i> third instar larvae (B). No staining is observed in the posterior compartment of discs that express <i>wg-RNAi</i>. (C) The frequency of RBF^D253A-induced ectopic tissue growth is not affected in <i>UAS-wg-RNAi</i> co-expressing flies (Chi² test, α = 0.15). All discs are shown with posterior to the top.</p

<i>RBF^D253A</i> expression induces proliferation of neighboring cells.

<p>(A–F) From left to right, phenotypes of the larvae are: <i>en-Gal4/+</i>, <i>en-Gal4/+</i>; <i>UAS-RBF/+</i> and <i>UAS-RBF^D253AX; en-Gal4/+</i>. (A–C) S phase staining by BrdU (green), and RBF immuno-staining (red). (D–F) BrdU staining (white) of the discs shown in (A–C). (B, E) In RBF expressing discs, as in the control disc shown in (A,D), BrdU staining is homogeneous in the whole disc, except in the ZNC (Zone of Non-proliferating Cells) (white arrow). (C–F) In RBF^D253A expressing discs, cells surrounding the strong RBF staining exhibit an enhanced BrdU staining, indicating that these cells have an increased proliferation rate. (G–L) From left to right, genotypes of the larvae are: <i>ptc-Gal4/+</i>, <i>ptc-Gal4/UAS-RBF</i>, and <i>UAS-RBF^D253A</i> X; <i>ptc-Gal4/+</i>. (G–I) S phase staining by BrdU (green), and RBF staining (red). (J–L) BrdU staining (white) of the discs shown in (G–I) with enlarged view of boxed area. (G, J) BrdU staining is homogeneous in the whole disc, except in the ZNC (white arrow). (I–L) In <i>ptc>RBF^D253A</i> discs, cells within the ZNC that are adjacent to RBF^D253A expressing cells are labeled with BrdU, indicating an abnormal proliferation of these cells. All discs are shown with posterior to the top.</p

A novel immunoregulatory role for NK-cell cytotoxicity in protection from HLH-like immunopathology in mice.

International audienceThe impairment of cytotoxic activity of lymphocytes disturbs immune surveillance and leads to the development of hemophagocytic lymphohistiocytic syndrome (HLH). Although cytotoxic T lymphocyte (CTL) control of HLH development is well documented, the role for natural killer (NK)-cell effector functions in the pathogenesis of this immune disorder remains unclear. In this study, we specifically targeted a defect in cytotoxicity to either CTL or NK cells in mice so as to dissect the contribution of these lymphocyte subsets to HLH-like disease severity after lymphocytic choriomeningitis virus (LCMV) infection. We found that NK-cell cytotoxicity was sufficient to protect mice from the fatal outcome that characterizes HLH-like disease and was also sufficient to reduce HLH-like manifestations. Mechanistically, NK-cell cytotoxicity reduced tissue infiltration by inflammatory macrophages and downmodulated LCMV-specific T-cell responses by limiting hyperactivation of CTL. Interestingly, the critical protective effect of NK cells on HLH was independent of interferon-γ secretion and changes in viral load. Therefore our findings identify a crucial role of NK-cell cytotoxicity in limiting HLH-like immunopathology, highlighting the important role of NK cytotoxic activity in immune homeostasis