Analysis of the role of the transcription factor Ikaros in plasmacytoid dendritic cells development

Le développement et la fonction des cellules dendritiques plasmacytoïdes (pDCs) doivent être finement régulés afin d’éviter le développement de maladies auto-immunes ou de leucémies. Il a été montré récemment qu’une fraction des leucémies humaines dérivées des pDCs possède des mutations de type perte de fonction dans le locus IKZF1 qui code pour le facteur de transcription Ikaros. Ainsi l’étude de la fonction d’Ikaros dans les pDCs pourrait aider à comprendre son possible rôle de gène suppresseur de tumeur. Les souris hypomorphiques pour Ikaros (IkL/L) ne possèdent pas de pDCs matures dans la rate et les ganglions lymphatiques mais accumulent des pDCs immatures dans la moelle osseuse (MO). De manièreintéressante, les pDCs de MO IkL/L montrent une activation ectopique de la voie Notch. Nous avons trouvé qu’un inhibiteur de gamma-secrétase (GSI), qui inhibe la voie Notch, permet de restaurer la différenciation de pDCs fonctionnelles dans les cultures de cellules de MO. Les principaux progéniteurs dendritiques affectés par le GSI sont le progéniteur myéloïde commun (CMP) et le progéniteur des macrophages et des cellules dendritiques (MDP). Comme le GSI inhibe la voie Notch, nous avons aussi inactivé RBPJ, le facteur de transcription situé en aval des récepteurs Notch. Contre toute attente, l’inactivation de RBPJ ne récapitule pas les effets observés avec le GSI. De plus, les cellules IkL/L déficientes pour RBPJ continuent de répondre au GSI, ce qui suggère que le GSI possède d’autres cibles en plus de la voie Notch dans ce système. Nos données montrent ainsi qu’Ikaros est requis pour la différenciation terminale des pDCs et qu’il agit en partie en bloquant une voie GSI dépendante Notch indépendante.The development and function of plasmacytoid dendritic cells (pDCs) must be tightly regulated to prevent autoimmune disease or leukemia. It was recently discovered that a fraction of human pDC-derived neoplasms exhibit loss of function mutations of the IKZF1 locus, which encodes the Ikaros transcription factor. Deciphering the function of Ikaros in pDCs could thus help understand its probable tumor suppressor function. Mice hypomorphic for Ikaros (IkL/L) are devoid of mature pDCs in the spleen and lymph nodes but accumulate immature pDCs in the bone marrow (BM). Interestingly IkL/L BM pDCs exhibit an ectopic activation of the Notch pathway. We found that a gamma secretase inhibitor (GSI), which inhibits Notch signalling,rescues the differentiation of functional pDCs in BM cultures. The main dendritic cell progenitors affected by GSI are the common myeloid progenitors (CMP) and the macrophage and dendritic cell progenitors (MDP). As GSI inhibits the activation of the Notch pathway, we also inactivated RBPJ, the transcriptional effector of the Notch pathway. Surprisingly, RBPJ inactivation did not recapitulate the effect of GSI. Moreover, RBPJdeficient IkL/L cells still respond to GSI, demonstrating that GSI targets additional events besides Notch in this system. Our data thus show that Ikaros is required for terminal differentiation of pDCs, and acts in part by blocking a Notch independent GSI-sensitive pathway

Analysis of the role of the transcription factor Ikaros in plasmacytoid dendritic cells development

Le développement et la fonction des cellules dendritiques plasmacytoïdes (pDCs) doivent être finement régulés afin d’éviter le développement de maladies auto-immunes ou de leucémies. Il a été montré récemment qu’une fraction des leucémies humaines dérivées des pDCs possède des mutations de type perte de fonction dans le locus IKZF1 qui code pour le facteur de transcription Ikaros. Ainsi l’étude de la fonction d’Ikaros dans les pDCs pourrait aider à comprendre son possible rôle de gène suppresseur de tumeur. Les souris hypomorphiques pour Ikaros (IkL/L) ne possèdent pas de pDCs matures dans la rate et les ganglions lymphatiques mais accumulent des pDCs immatures dans la moelle osseuse (MO). De manièreintéressante, les pDCs de MO IkL/L montrent une activation ectopique de la voie Notch. Nous avons trouvé qu’un inhibiteur de gamma-secrétase (GSI), qui inhibe la voie Notch, permet de restaurer la différenciation de pDCs fonctionnelles dans les cultures de cellules de MO. Les principaux progéniteurs dendritiques affectés par le GSI sont le progéniteur myéloïde commun (CMP) et le progéniteur des macrophages et des cellules dendritiques (MDP). Comme le GSI inhibe la voie Notch, nous avons aussi inactivé RBPJ, le facteur de transcription situé en aval des récepteurs Notch. Contre toute attente, l’inactivation de RBPJ ne récapitule pas les effets observés avec le GSI. De plus, les cellules IkL/L déficientes pour RBPJ continuent de répondre au GSI, ce qui suggère que le GSI possède d’autres cibles en plus de la voie Notch dans ce système. Nos données montrent ainsi qu’Ikaros est requis pour la différenciation terminale des pDCs et qu’il agit en partie en bloquant une voie GSI dépendante Notch indépendante.The development and function of plasmacytoid dendritic cells (pDCs) must be tightly regulated to prevent autoimmune disease or leukemia. It was recently discovered that a fraction of human pDC-derived neoplasms exhibit loss of function mutations of the IKZF1 locus, which encodes the Ikaros transcription factor. Deciphering the function of Ikaros in pDCs could thus help understand its probable tumor suppressor function. Mice hypomorphic for Ikaros (IkL/L) are devoid of mature pDCs in the spleen and lymph nodes but accumulate immature pDCs in the bone marrow (BM). Interestingly IkL/L BM pDCs exhibit an ectopic activation of the Notch pathway. We found that a gamma secretase inhibitor (GSI), which inhibits Notch signalling,rescues the differentiation of functional pDCs in BM cultures. The main dendritic cell progenitors affected by GSI are the common myeloid progenitors (CMP) and the macrophage and dendritic cell progenitors (MDP). As GSI inhibits the activation of the Notch pathway, we also inactivated RBPJ, the transcriptional effector of the Notch pathway. Surprisingly, RBPJ inactivation did not recapitulate the effect of GSI. Moreover, RBPJdeficient IkL/L cells still respond to GSI, demonstrating that GSI targets additional events besides Notch in this system. Our data thus show that Ikaros is required for terminal differentiation of pDCs, and acts in part by blocking a Notch independent GSI-sensitive pathway

Ikaros cooperates with Notch activation and antagonizes TGFβ signaling to promote pDC development

International audiencePlasmacytoid and conventional dendritic cells (pDCs and cDCs) arise from monocyte and dendritic progenitors (MDPs) and common dendritic progenitors (CDPs) through gene expression changes that remain partially understood. Here we show that the Ikaros transcription factor is required for DC development at multiple stages. Ikaros cooperates with Notch pathway activation to maintain the homeostasis of MDPs and CDPs. Ikaros then antagonizes TGFβ function to promote pDC differentiation from CDPs. Strikingly, Ikaros-deficient CDPs and pDCs express a cDC-like transcriptional signature that is correlated with TGFβ activation, suggesting that Ikaros is an upstream negative regulator of the TGFβ pathway and a repressor of cDC-lineage genes in pDCs. Almost all of these phenotypes can be rescued by short-term in vitro treatment with γ-secretase inhibitors, which affects both TGFβ-dependent and -independent pathways, but is Notch-independent. We conclude that Ikaros is a crucial differentiation factor in early dendritic progenitors that is required for pDC identity

Genetic deletion of RBPJ does not rescue Ik^L/L pDC differentiation.

<p><b>(A)</b> Representative analysis of Flt3L-supplemented cultures of BM cells from compound mutant mice with Ik^L/L and/or RBPJ KO alleles, after addition of DMSO or GSI at day 0. Cultures were analyzed 8d later. <b>(B)</b> Numbers of total cells and pDCs obtained from cultures described in (A) (mean±SD from 3–5 mice per genotype; p values were obtained by paired Student’s t-test). <b>(C)</b> Analysis and <b>(D)</b> relative numbers of MDPs and CDPs from the BM of Ikaros-RBPJ compound mutant mice (representative of 2 independent experiments with 2–5 mice per genotype and per experiment; p values were obtained by Student’s t-test). *p≤0.05; **p≤0.01; ***p≤0.001.</p

The TGFβ1 pathway is activated in Ik^L/L CDPs.

<p>CD45.2⁺ WT or Ik^L/L MDPs, and CDPs, were co-cultured with supporting CD45.1⁺ WT BM cells for 24h with Flt3L and GSI (or DMSO). CD45.2⁺ cells were then re-purified and their transcriptomes analyzed. 2 mice per condition. <b>(A)</b> Heat map representing K-means clustering of 963 genes differentially expressed between WT or Ik^L/L CDPs [fold change (FC)>1.5]. Clusters I and II are deregulated specifically in Ik^L/L CDPs. Clusters III and IV are deregulated in all Ik^L/L DC progenitors. Eng indicates the Endoglin gene. Red and green indicate high and low expression, respectively. <b>(B)</b> Hierarchical clustering of the genes from clusters II and IV in (A), using Immgen transcriptome data for DC progenitors and mature subsets (GSE15907). Clusters of genes similarly expressed between Ik^L/L CDPs and DC progenitors (Imm) or mature DCs (DC) are indicated. <b>(C)</b> K-means clustering of 70 genes differentially expressed between WT and Ik^L/L CDPs, and deregulated by GSI (FC>1.5). <b>(D)</b> Top 5 putative upstream regulators related to the 70 genes from (C), as identified by the Ingenuity Pathways Analysis software. <b>(E)</b> GSEA enrichment plots of genes specifically down-regulated [clusters I and III in (A)] and <b>(F)</b> up-regulated (clusters II and IV) in Ik^L/L CDPs. The ranked gene list corresponds to TGFβ1-regulated genes in CDPs, as identified by Felker et al (2010). NES: normalized enrichment score; FDR: false discovery rate. <b>(G)</b> Genome browser tracks showing Ikaros binding to loci associated with TGFβ activation in pre-B cells (BH1-Ik1-ER-Bcl2 cell line) and immature DN3 thymocytes (GEO GSE114629 and GSE61148 accession numbers).</p

Notch pathway activation in Ik^L/L pDCs.

<p><b>(A)</b><i>Hes1</i> and <i>Ptcra</i> mRNA expression in BM pDCs from 4 WT and 5 (Ik^L/L) mice, as analyzed by RT-qPCR and normalized to <i>Hprt</i> mRNA levels (mean±SD of triplicate data). (<b>B)</b> GFP reporter expression (black line) in total BM cells and BM pDCs (CD11c⁺CD317⁺) from Hes1-GFP⁺ WT and Ik^L/L mice, by flow cytometry. Grey histograms correspond to control cells from mice lacking the Hes1-GFP reporter. <b>(C)</b> Percentage of GFP⁺ BM pDCs (CD11c⁺CD317⁺) from Hes1-GFP⁺ WT and Ik^L/L mice, as analyzed in (B). *p≤0.05 (Student’s t-test). <b>(D)</b> CCR9 and SiglecH vs. GFP expression in BM pDCs from Hes1-GFP⁺ WT and Ik^L/L mice. Representative of 3 independent experiments.</p

Ikaros regulates DC progenitor development.

<p><b>(A)</b> Representative analysis of MDPs, CDPs, <b>(B)</b> pre-cDCs, and <b>(C)</b> pDCs from Ik^L/L (L/L) and WT BM, by flow cytometry. <b>(D)</b> Representative analysis of splenic cDCs (CD11c⁺MHCII⁺). <b>(E)</b> Relative numbers of BM MDPs, CDPs and pre-cDCs (as gated in A and B), <b>(F)</b> BM pDCs (as gated in C), and <b>(G)</b> splenic cDCs (as gated in D). Mean±SD of 4–12 animals per group. *p≤0.05; **p≤0.01; ***p≤0.001 (Student’s t-test).</p

Inhibition of γ-secretase rescues Ik^L/L pDC differentiation in vitro.

<p><b>(A)</b> Percentage of CD11c⁺CD317⁺CD11b^- pDCs after 8 days of Flt3L-supplemented WT and Ik^L/L BM cultures, treated with GSI or vehicle (DMSO) at the indicated days of culture. Representative of >5 independent experiments. <b>(B)</b> Percentage of pDCs from Flt3L-supplemented WT and Ik^L/L BM cultures, treated with GSI or DMSO at day 0 and analyzed at day 8. <b>(C, D)</b> Numbers of pDCs (C) and total cell numbers (D) obtained from cultures described in (B). *p≤0.05; **p≤0.01; ***p≤0.001 (Student’s t-test). <b>(E)</b> CCR9, Ly49Q and B220 expression on pDCs cultured as in (B). Representative of 3 independent experiments. <b>(F)</b> RT-qPCR analysis of <i>Ifna</i> expression induced from pDCs after in vitro culture. WT and Ik^L/L pDCs were sorted at d8 of culture, after GSI treatment at day 0, and stimulated for 16h with CpG ODN 1585. <i>Ifna</i> mRNA levels were measured by RT-qPCR and normalized to <i>Ubb</i> mRNA. nd: not done.</p

TGFβ1 activation inhibits pDC development from Ik^L/L CDPs.

<p><b>(A)</b> CD105 expression on WT and Ik^L/L DC progenitors. <b>(B)</b> SiglecH vs. CD105 expression on BM pDCs (CD317⁺) and non-pDCs (CD317^-). Representative of 4 independent experiments. <b>(C)</b> Effect of the TGFβR1 inhibitor SB431542 on pDC differentiation in Flt3L-supplemented WT and Ik^L/L BM cultures. Cells were treated at d0 with SB431542 and/or GSI and analyzed at d8. Percentages of cells in the corresponding gates are indicated. Representative of 4 independent experiments. <b>(D)</b> Number of pDCs obtained from experiments described in (C). Data of the SB431542 treatments are shown at a larger scale in the lower panel. Representative of 4 independent experiments; 2 mice per genotype per experiment; p values were obtained with a Student’s t-test. *p≤0.05. <b>(E)</b> Schematic representation of Ikaros function during DC development in the BM. Ikaros and Notch signaling are required for the onset of DC differentiation and the appearance of MDPs and CDPs. Later, in CDPs, Ikaros promotes pDC development by antagonizing TGFβ1 signaling and by repressing the cDC gene expression program. HSPC: Hematopoietic stem/progenitor cell.</p

Identification of monocyte-like precursors of granulocytes in cancer as a mechanism for accumulation of PMN-MDSCs.

We have identified a precursor that differentiates into granulocytes in vitro and in vivo yet belongs to the monocytic lineage. We have termed these cells monocyte-like precursors of granulocytes (MLPGs). Under steady state conditions, MLPGs were absent in the spleen and barely detectable in the bone marrow (BM). In contrast, these cells significantly expanded in tumor-bearing mice and differentiated to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Selective depletion of monocytic cells had no effect on the number of granulocytes in naive mice but decreased the population of PMN-MDSCs in tumor-bearing mice by 50%. The expansion of MLPGs was found to be controlled by the down-regulation of Rb1, but not IRF8, which is known to regulate the expansion of PMN-MDSCs from classic granulocyte precursors. In cancer patients, putative MLPGs were found within the population of CXCR1+CD15-CD14+HLA-DR-/lo monocytic cells. These findings describe a mechanism of abnormal myelopoiesis in cancer and suggest potential new approaches for selective targeting of MDSCs. © 2019 Mastio et al