Dominique Bonnet

Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8α+ dendritic cells

In mouse, a subset of dendritic cells (DCs) known as CD8α+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However, translation into clinical protocols has been hampered by the failure to identify CD8α+ DCs in humans. Here, we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8α+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8α+ DCs, human DNGR-1+ BDCA3hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8α+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8, but not of TLR7, and produce interleukin (IL)-12 when given innate and T cell–derived signals. Notably, DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy.

Expansion of human SCID-repopulating cells under hypoxic conditions

It has been proposed that bone marrow (BM) hematopoietic stem and progenitor cells are distributed along an oxygen (O2) gradient, where stem cells reside in the most hypoxic areas and proliferating progenitors are found in O2-rich areas. However, the effects of hypoxia on human hematopoietic stem cells (HSCs) have not been characterized. Our objective was to evaluate the functional and molecular responses of human BM progenitors and stem cells to hypoxic conditions. BM lineage-negative (Lin-) CD34+CD38- cells were cultured in serum-free medium under 1.5% O2 (hypoxia) or 20% O2 (normoxia) for 4 days. Using limiting dilution analysis, we demonstrate that the absolute number of SCID-repopulating cells (SRCs) increased by 5.8-fold in hypoxic cultures compared with normoxia, and by 4.2-fold compared with freshly isolated Lin-CD34+CD38- cells. The observed increase in BM-repopulating activity was associated with a preferential expansion of Lin-CD34+CD38- cells. We also demonstrate that, in response to hypoxia, hypoxia-inducible factor-1alpha protein was stabilized, surface expression of angiogenic receptors was upregulated, and VEGF secretion increased in BM Lin-CD34+ cultures. The use of low O2 levels to enhance the survival and/or self-renewal of human BM HSCs in vitro represents an important advance and could have valuable clinical implications.

Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34− fraction

Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34(+) fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34(+) fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34(-) fraction, whereas the CD34(+) fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34(+) and CD34(-) fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.

Chimeric Antigen Receptors against CD33/CD123 antigens efficiently target primary Acute Myeloid Leukemia cells in vivo

As significant numbers of acute myeloid leukemia (AML) patients are still refractory to conventional therapies or experience relapse, immunotherapy using T cells expressing chimeric antigen receptors (CARs) might represent a valid treatment option. AML cells frequently overexpress the myeloid antigens CD33 and CD123, for which specific CARs can be generated. However, CD33 is also expressed on normal hematopoietic stem/progenitor cells (HSPCs), and its targeting could potentially impair normal hematopoiesis. In contrast, CD123 is widely expressed by AML, while low expression is detected on HSPCs, making it a much more attractive target. In this study we describe the in vivo efficacy and safety of using cytokine-induced killer (CIK) cells genetically modified to express anti-CD33 or anti-CD123 CAR to target AML. We show that both these modified T cells are very efficient in reducing leukemia burden in vivo, but only the anti-CD123 CAR has limited killing on normal HSPCs, thus making it a very attractive immunotherapeutic tool for AML treatment.

Small Neutralizing Molecules to Inhibit Actions of the Chemokine CXCL12

The chemokine CXCL12 and the receptor CXCR4 play pivotal roles in normal vascular and neuronal development, in inflammatory responses, and in infectious diseases and cancer. For instance, CXCL12 has been shown to mediate human immunodeficiency virus-induced neurotoxicity, proliferative retinopathy and chronic inflammation, whereas its receptor CXCR4 is involved in human immunodeficiency virus infection, cancer metastasis and in the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome. As we screened chemical libraries to find inhibitors of the interaction between CXCL12 and the receptor CXCR4, we identified synthetic compounds from the family of chalcones that reduce binding of CXCL12 to CXCR4, inhibit calcium responses mediated by the receptor, and prevent CXCR4 internalization in response to CXCL12. We found that the chemical compounds display an original mechanism of action as they bind to the chemokine but not to CXCR4. The highest affinity molecule blocked chemotaxis of human peripheral blood lymphocytes ex vivo. It was also active in vivo in a mouse model of allergic eosinophilic airway inflammation in which we detected inhibition of the inflammatory infiltrate. The compound showed selectivity for CXCL12 and not for CCL5 and CXCL8 chemokines and blocked CXCL12 binding to its second receptor, CXCR7. By analogy to the effect of neutralizing antibodies, this molecule behaves as a small organic neutralizing compound that may prove to have valuable pharmacological and therapeutic potential.

Identification and pharmacological properties of E339-3D6, the first nonpeptidic apelin receptor agonist

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To explore further upstream the role played by this peptide, nonpeptidic agonists and antagonists of the apelin receptor are required. To identify such compounds that do not exist to date, we used an original fluorescence resonance energy transfer‐based assay to screen a G‐protein‐coupled receptor‐focused library of fluorescent compounds on the human EGFP‐tagged apelin receptor. This led to isolated E339–3D6 that displayed a 90 nM affinity and behaved as a partial agonist with regard to cAMP production and as a full agonist with regard to apelin receptor internalization. Finally, E339–3D6 induced vasorelaxation of rat aorta precontracted with noradrenaline and potently inhibited systemic vasopressin release in water‐deprived mice when intracerebroventricularly injected. This compound represents the first nonpeptidic agonist of the apelin receptor, the optimization of which will allow development of a new generation of vasodilator and aquaretic agents.—Iturrioz, X., Alvear‐Perez, R., De Mota, N., Franchet, C., Guillier, F., Leroux, V., Dabire, H., Le Jouan, M., Chabane, H., Gerbier, R., Bonnet, D., Berdeaux, A., Maigret, B., Galzi J.‐L., Hibert, M., Llorens‐Cortes, C. Identification and pharmacological properties of E339–3D6, FASEB J. 24, 1506–1517 (2010). www.fasebj.org

T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments

It is widely accepted that complex interactions between cancer cells and their surrounding microenvironment contribute to disease development, chemo-resistance and disease relapse. In light of this observed interdependency, novel therapeutic interventions that target specific cancer stroma cell lineages and their interactions are being sought. Here we studied a mouse model of human T-cell acute lymphoblastic leukaemia (T-ALL) and used intravital microscopy to monitor the progression of disease within the bone marrow at both the tissue-wide and single-cell level over time, from bone marrow seeding to development/selection of chemo-resistance. We observed highly dynamic cellular interactions and promiscuous distribution of leukaemia cells that migrated across the bone marrow, without showing any preferential association with bone marrow sub-compartments. Unexpectedly, this behaviour was maintained throughout disease development, from the earliest bone marrow seeding to response and resistance to chemotherapy. Our results reveal that T-ALL cells do not depend on specific bone marrow microenvironments for propagation of disease, nor for the selection of chemo-resistant clones, suggesting that a stochastic mechanism underlies these processes. Yet, although T-ALL infiltration and progression are independent of the stroma, accumulated disease burden leads to rapid, selective remodelling of the endosteal space, resulting in a complete loss of mature osteoblastic cells while perivascular cells are maintained. This outcome leads to a shift in the balance of endogenous bone marrow stroma, towards a composition associated with less efficient haematopoietic stem cell function. This novel, dynamic analysis of T-ALL interactions with the bone marrow microenvironment in vivo, supported by evidence from human T-ALL samples, highlights that future therapeutic interventions should target the migration and promiscuous interactions of cancer cells with the surrounding microenvironment, rather than specific bone marrow stroma, to combat the invasion by and survival of chemo-resistant T-ALL cells.

Biased Agonist Pharmacochaperones of the AVP V2 Receptor May Treat Congenital Nephrogenic Diabetes Insipidus

X-linked congenital nephrogenic diabetes insipidus (cNDI) results from inactivating mutations of the human arginine vasopressin (AVP) V2 receptor (hV(2)R). Most of these mutations lead to intracellular retention of the hV(2)R, preventing its interaction with AVP and thereby limiting water reabsorption and concentration of urine. Because the majority of cNDI-hV(2)Rs exhibit protein misfolding, molecular chaperones hold promise as therapeutic agents; therefore, we sought to identify pharmacochaperones for hV(2)R that also acted as agonists. Here, we describe high-affinity nonpeptide compounds that promoted maturation and membrane rescue of L44P, A294P, and R337X cNDI mutants and restored a functional AVP-dependent cAMP signal. Contrary to pharmacochaperone antagonists, these compounds directly activated a cAMP signal upon binding to several cNDI mutants. In addition, these molecules displayed original functionally selective properties (biased agonism) toward the hV(2)R, being unable to recruit arrestin, trigger receptor internalization, or stimulate mitogen-activated protein kinases. These characteristics make these hV(2)R agonist pharmacochaperones promising therapeutic candidates for cNDI.

Proper desensitization of CXCR4 is required for lymphocyte development and peripheral compartmentalization in mice

Desensitization controls G protein-dependent signaling of chemokine receptors. We investigate the physiologic implication of this process for CXCR4 in a mouse model harboring a heterozygous mutation of the Cxcr4 gene, which engenders a desensitization-resistant receptor. Such anomaly is linked to the warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, a human rare combined immunodeficiency. Cxcr4(+/mutant(1013)) mice display leukocytes with enhanced responses to Cxcl12 and exhibit leukopenia as reported in patients. Treatment with CXCL12/CXCR4 antagonists transiently reverses blood anomalies, further demonstrating the causal role of the mutant receptor in the leukopenia. Strikingly, neutropenia occurs in a context of normal bone marrow architecture and granulocyte lineage maturation, indicating a minor role for Cxcr4-dependent signaling in those processes. In contrast, Cxcr4(+/1013) mice show defective thymopoiesis and B-cell development, accounting for circulating lymphopenia. Concomitantly, mature T and B cells are abnormally compartmentalized in the periphery, with a reduction of primary follicles in the spleen and their absence in lymph nodes mirrored by an unfurling of the T-cell zone. These mice provide a model to decipher the role of CXCR4 desensitization in the homeostasis of B and T cells and to investigate which manifestations of patients with WHIM syndrome may be overcome by dampening the gain of CXCR4 function.

α5β1 integrin antagonists reduce chemotherapy-induced premature senescence and facilitate apoptosis in human glioblastoma cells

The α5β1 integrin represent a new therapeutic target for glioblastoma, which are malignant brain tumors difficult to cure with conventional therapies. Glioblastoma are known to be highly resistant to chemotherapy. We, therefore, investigated whether blocking α5β1 integrin with specific nonpeptidic antagonists concomitantly with chemotherapy (ellipticine and temozolomide) may impact the response to chemotherapy of human glioblastoma. Here we show that inhibiting α5β1 integrin with 2 selective ligands (SJ749 and K34c) decreases chemotherapy‐induced premature senescence and facilitates cell apoptosis in a functional p53 background (U87MG cells). When p53 is mutated and inactive (U373 cells), chemotherapy induces p53‐independent cell apoptosis instead of senescence that is not improved by integrin antagonists. Silencing p53 in U87MG cells with siRNA as well as evaluating HCT116 p53+/+ and p53−/− colon carcinoma cell behavior support the hypothesis of an as yet unknown effect of α5β1 integrin antagonists on the control of chemotherapy‐induced premature senescence and apoptosis. α5β1 integrin antagonists modulate the p53 signaling induced by chemotherapy. Our results highlight a new role of the α5β1 integrin in the control of glioblastoma aggressiveness and responsiveness to chemotherapy, which may have a crucial impact in the clinical management of patients suffering from brain tumors.