|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 15 December 2006, Vol. 108, No. 13, pp. 4025-4034. Prepublished online as a Blood First Edition Paper on August 15, 2006; DOI 10.1182/blood-2006-03-007757.
HEMATOPOIESIS Ikaros is required for plasmacytoid dendritic cell differentiationFrom the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia; the Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Marseille, France; Institut National de la Santé et de la Recherche Médicale (INSERM) U631, Marseille, France; Centre National de la Recherche Scientifique (CNRS) Unité mixte de recherche (UMR) 6102, Marseille, France; Schering-Plough Research Institute, Laboratory for Immunological Research, Dardilly, France; the Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Brown University, Providence, RI; and Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP (Université Louis Pasteur), Illkirch, Communauté Urbaine (CU), Strasbourg, France.
Plasmacytoid dendritic cells (pDCs) are specialized DCs that produce high levels of type I IFN upon viral infection. Despite their key immunoregulatory role, little is known about pDC ontogeny or how developmental events regulate their function. We show that mice expressing low levels of the transcription factor Ikaros (IkL/L) lack peripheral pDCs, but not other DC subsets. Loss of pDCs is associated with an inability to produce type I IFN after challenge with Toll-like receptor-7 and -9 ligands, or murine cytomegalovirus (MCMV) infection. In contrast, conventional DCs are present in normal numbers and exhibit normal responses in vivo after challenge with MCMV or inactivated toxoplasma antigen. Interestingly, IkL/L bone marrow (BM) cells contain a pDC population that appears blocked at the Ly-49Q– stage of differentiation and fails to terminally differentiate in response to Flt-3L, a cytokine required for pDC differentiation. This differentiation block is strictly dependent on a cell-intrinsic requirement for Ikaros in pDC-committed precursors. Global gene expression profiling of IkL/L BM pDCs reveals an up-regulation of genes not normally expressed, or expressed at low levels, in WT pDCs. These studies suggest that Ikaros controls pDC differentiation by silencing a large array of genes.
Plasmacytoid dendritic cells (pDCs) are a unique DC subset. pDCs produce large quantities of type I IFN (  ,  ,or  ) in response to viral infection and bacterial components,1-5 and thus are considered key cells for the immune response to these pathogens. Human and mouse pDCs have been implicated in the activation of natural killer (NK) cells, the differentiation of regulatory T cells and plasma cells, as well as in the polarization of naive T cells into Th1/Th2 responders.6-14 These pleiotropic roles highlight the importance of pDCs in the control of T-cell tolerance, graft-versus-host disease, allergic responses, and lupus erythematosus.15-20 Despite numerous studies, however, pDC function in vivo remains incompletely understood.
Similarly, little is known about the specific signals and factors involved in pDC differentiation.21 Flt-3 signaling has been described as a crucial component of the pDC developmental program.22-24 Recent studies have shown that pDCs develop from Flt-3+ cells within the common lymphoid or common myeloid progenitor populations in mouse bone marrow.25,26 Since many pDCs express some lymphoid-related gene products (such as pT
Several transcription factors have been identified that regulate the development of distinct DC types.28-31 Among these is Ikaros, a zinc finger protein essential for the development of multiple hematopoietic lineages.32-36 Ikaros functions mainly as a repressor.37-39 It binds DNA as homodimers or heterodimers with other members of the Ikaros family such as Aiolos, expressed mainly in B and T cells, and Helios, expressed in early hematopoietic precursors and T cells.40-43 Expression of a dominant-negative form of Ikaros results in a complete loss of all conventional DC (cDC) subsets, while a null mutation in Ikaros leads to the selective loss of CD11c+CD11b+ but not CD11c+CD8
Our laboratory has described a mutant mouse line carrying a hypomorphic mutation in the Ikaros locus (IkL/L) in which a
Mice The IkL/L mouse line was previously described.35 Mice (6-10 weeks old) used in this study were maintained under specific pathogen-free (SPF) conditions, and were backcrossed more than 7 generations onto the C57Bl/6 genetic background. Most experiments used mice from a 10th backcross generation. All gave similar results. B6.Ly5SJL congenics were also maintained under SPF conditions. Antibodies and flow cytometry All antibodies were from BD Pharmingen (San Diego, CA) or eBiosciences (San Diego, CA) except for biotin-conjugated Ly-6C (clone ER-MP20; BMA Biomedicals, Augst, Switzerland) and anti–Ly-49Q (MBL, Watertown, MA). Alexa488-conjugated 120G8 was described previously.45 CD11c (N418), CD11b (M1/70), and B220 (RA3-6B2) were purified and conjugated to FITC, PE, allophycocyanin (APC), or biotin according to standard protocols. For fluorescence-activated cell sorting (FACS) analyses, cells were first incubated with anti-CD16/32 to block Fc receptors. Intracellular IL-12 was detected according to Dalod et al.46 Cells were analyzed on a FACSCalibur or LSR II (BD BioSciences, San Jose, CA). Results were analyzed with the FlowJo software (TreeStar, Ashland, OR). Sorting was performed on a FACSVantage SE option DiVa (BD BioSciences). Cell preparations Organs were digested by collagenase (liberase CI; Boehringer Mannheim, Mannheim, Germany) as described.47 Cell suspensions were treated with 0.165 MNH4Cl to lyse red blood cells (RBCs). For experiments requiring DC enrichment from the spleen, cells were incubated with anti-CD3 (17A2) and anti-CD19, followed by goat anti–rat IgG–coated Dynabeads (Dynal, Lake Success, NY), and depleted with a Dynal magnet. Depleted cells were then positively purified for CD11c+ cells using CD11c+ Microbeads and MiniMacs (Miltenyi Biotec, Auburn, CA). Purity was more than 95%. For enrichment from the BM, WT cells were depleted of CD19+ cells by the Dynal method, then purified with the indicated antibodies by FACS. IkL/L BM cells were not depleted but were similarly purified. Sort purity was more than 95%. In vitro stimulation
Cells were cultured in RPMI 1640 (Life Technologies, Bethesda, MD), 10% FCS (Life Technologies), 2 mM L-glutamine, 10 mM HEPES, 50 µM 2-ME, and 80 µg/mL gentamycin. Cells were stimulated at 105 cells/well in 200 µL in 96-well plates. Poly I:C (Invitrogen, Frederick, MD) was used at 20 µg/mL final concentration. The formaldehyde-inactivated human influenza virus, strain NK/TM/138/00 (a kind gift from N. Kuehn, Aventis Pasteur, France), was used at 100 hemagglutinin U/mL. CpG D19 (GGT GCA TCG ATG CAG GGG GG) was phosphorothioate-modified (MWG Biotec, High Point, NC) and used at 10 µg/mL. Murine recombinant Flt-3L (25 ng/mL; R&D Systems, Minneapolis, MN) was used as previously described.23 Supernatants were collected after 24 hours of culture and tested for IFN In vivo treatments
Mice were anesthetized and injected intravenously in the retro-orbital vein with 200 µL CpG 1668 (TCC ATG ACG TTC CTG ATG CT), prepared as described.2 R848 (Invivogen, San Diego, CA) was used at 5 µg/mouse in PBS. Poly I:C (Invitrogen) was used at 50 µg/mouse in PBS. Control mice were injected with PBS. Sera were analyzed using the ELISAs described for in vitro stimulation. Infections were initiated with 2 x 104 to 5 x 104 plaque-forming units of a salivary gland–extracted GFP-recombinant murine cytomegalovirus (MCMV) Smith strain (RVG-102) injected intraperitoneally on day 0. Control mice were injected with the medium in which the virus stocks were diluted. At 1.5 days after challenge, frequencies of splenic pDCs, MCMV-infected DCs, and IL-12+ DCs were analyzed as described. IFN BM chimeras B6.Ly5SJL congenics were irradiated (9 gray) 24 hours before reconstitution. WT (2 x 106) or IkL/L (5 x 106) BM cells, both expressing the Ly5B6 allele, were harvested from donor mice and injected intravenously into the recipients. For the double chimera experiments, WT or IkL/L BM cells were mixed with freshly-isolated, nonirradiated B6.Ly5SJL cells before injection, at a 1:1 ratio for WT cells (1 x 106:1 x 106) and a 5:1 ratio for IkL/L cells (5 x 106:1 x 106). Ly5B6 cells were analyzed 6 to 10 weeks after transfer using an anti-Ly5B6 (Ly5.2) allele-specific antibody (clone 104). Reconstitution efficiency ranged between 30% and 50% for IkL/L and between 50% and 70% for WT donor cells. RT-PCR
Total RNA was extracted from sorted populations (2 x 105 cells) using Trizol (Invitrogen) and reverse transcribed using AMV reverse transcriptase. Reversetranscription–polymerase chain reaction (RT-PCR) was performed with cDNA from the equivalent of 2 x 104 cells with the following primers: Microarray experiments and analysis Three WT and 3 IkL/L samples of 2 x 105 CD11c+120G8+ BM cells were sorted and RNA extracted with the Qiagen micro RNAeasy kit (Valencia, CA), yielding approximately 30 ng total RNA for each sample. Quality and absence of genomic DNA contamination was assessed with a Bioanalyser (Agilent, Palo Alto, CA). Probes were synthesized using 2 successive rounds of cRNA amplification, according to standard Affymetrix protocols, and hybridized to mouse 430 2.0 chips (Affymetrix, Santa Clara, CA). Raw data were transformed with the Mas5 algorithm, which yields a normalized expression value, and "absent" and "present" calls. Target intensity was set to 100 for all chips. Using Mas5, we performed all possible pairwise analyses, which yield "increased" (I), "decreased" (D), "marginally increased," "marginally decreased," or "not changed" calls, as well as fold change values. To select probe sets specifically increased or decreased in IkL/L samples compared with WT, we imposed that an I or D call be met by all possible IkL/L/WT pairwise comparisons. To evaluate the number of changes that might be due to experimental or biologic noise, we performed similar analyses, comparing "nonspecific" groups of 3 samples in which given WT and mutant samples had been permuted between groups (eg, comparing a first group containing 2 WT and 1 IkL/L samples with a second group containing 2 IkL/L and 1 WT samples). All 9 possible nonspecific combinations were analyzed. In 6 combinations, no probe sets were increased or decreased in all pairwise comparisons. In 3 combinations, fewer than 15 genes exhibited variations.
Selective loss of peripheral pDCs in mice with reduced Ikaros activity
As DC development has been shown to be affected upon loss of Ikaros activity,30,44 we examined distinct DC subpopulations in the spleens of WT, heterozygote Ik+/L, and homozygote IkL/L mice, using a combination of antibodies including the pDC-specific antibody, 120G8.45 WT, Ik+/L, and IkL/L spleens contained similar proportions of "myeloid" (CD11c+CD11b+) and "lymphoid" (CD11c+CD8
To determine if pDCs are functionally lacking in these animals, we used both in vitro and in vivo assays to test the capacity of IkL/L mice to produce IFN in response to stimuli known to activate pDCs through the triggering of Toll-like receptors (TLRs). The TLR ligands included influenza virus, TLR9-binding CpG oligonucleotides, and the TLR7 ligand R848, all of which can also induce both pDCs and cDCs to produce IL-12.1,48 As shown in Figure 2A, when CD11c+ WT and IkL/L spleen cells were cultured with influenza virus or CpG D19, WT cells produced IFN but IkL/L cells did not. Likewise, injection of CpG 1668 or R848 induced IFN production in WT but not mutant animals (Figure 2B). In contrast, normal IL-12 production was observed in all conditions, suggesting that TLR and/or cDC responses were not grossly altered in IkL/L mice. The in vivo IFN response to polyinosinic: polycytidylic acid (poly I:C), which activates a variety of cell types through TLR3,49 was also unaffected in IkL/L mice. Thus, IkL/L mice specifically lack pDCs, but not cDCs, both at the phenotypic and functional levels.
Impaired pDC response to MCMV infection
The lack of a peripheral pDC compartment implies that IkL/L mice may be susceptible to systemic infections. As pDCs have been reported to be the major IFN
While WT mice produced robust levels of IFN
In vivo IkL/L cDC responses are normal
To further confirm that IkL/L cDCs function normally in vivo, we tested their capacity to produce IL-12 and up-regulate maturation markers in 2 systems—MCMV infection and stimulation with Toxoplasma gondii soluble tachyzoite antigen (STAg).51 MCMV infection induces both CD8
A pDC population in the IkL/L bone marrow
As pDC development normally occurs in the BM, we examined the IkL/L BM for pDC differentiation. WT BM contained a population of CD11c+B220+Ly-6C+120G8+ cells that was similar in phenotype and frequency to splenic pDCs, although B220 expression was more heterogeneous in the BM population (Figure 4A; Table 1). Surprisingly, 120G8+ cells were also detected in similar numbers in the IkL/L BM; these cells expressed low levels of CD11c and were positive for Ly-6C, but negative for B220. Loss of the B220 marker was not a general phenomenon in IkL/L mice, as it is expressed at normal levels by IkL/L B cells.35 We compared the expression of other pDC-related markers on IkL/L BM 120G8+ cells and WT BM and splenic pDCs (Figure 4B). Notably, a larger proportion of IkL/L 120G8+ cells expressed high levels of CD8
A hallmark of pDC identity is the capacity to produce IFN
BM pDC development can be enhanced by Flt-3/Flt-3 ligand (Flt-3L) interactions,22-24 and Ikaros appears to be required for Flt-3 expression at the mRNA level in hematopoietic progenitors.34 We therefore asked if IkL/L BM 120G8+ cells were blocked in their differentiation due to a loss of Flt-3 signaling. To address this issue, we analyzed Flt-3 expression, and responsiveness to Flt-3L in IkL/L BM 120G8+ cells. IkL/L 120G8+ BM cells expressed strong levels of surface Flt-3 (Figure 5A), which at times were higher than WT levels, depending on the mouse (not shown). Importantly, culture of unfractionated BM cells with Flt-3L led to the ready emergence of CD11c+120G8+ cells in both WT and IkL/L cultures, with similar frequency and kinetics (Figure 5B-C). These results indicate that Flt-3 signaling is functional in IkL/L pDC precursors, even though the cells derived from IkL/L BM still failed to up-regulate B220.
Together, our results show that a pDC population resides in the bone marrow of IkL/L mice. These cells produce IFN The pDC defect is intrinsic to IkL/L pDC progenitor cells As Ikaros is expressed by all hematopoietic cells, it was important to distinguish if the block in pDC development in IkL/L mice was due to a cell-intrinsic defect within the pDC lineage or to an indirect effect due to diminished Ikaros levels in another hematopoietic population. To allow the development of IkL/L BM cells in a WT environment, we established single and double (mixed) bone marrow chimeras. In the single chimeras, we used unfractionated WT or IkL/L BM cells (both expressing the Ly5B6 allele) to reconstitute lethally irradiated B6.Ly5SJL recipients. These experiments tested the capacity of IkL/L hematopoietic progenitors to differentiate in an environment containing WT stromal cells. In the double chimeras, we used WT or IkL/L BM cells mixed with B6.Ly5SJL congenic BM cells to reconstitute irradiated B6.Ly5SJL recipients. This latter combination tested the capacity of IkL/L hematopoietic progenitors to differentiate in an environment containing both WT stromal and hematopoietic elements. Double chimeras also allowed us to determine if IkL/L hematopoietic cells can interfere with WT pDC development, or reciprocally, if the presence of WT (B6.Ly5SJL) BM cells can rescue IkL/L pDC development. Ly5B6 spleen and BM cells were analyzed 6 to 10 weeks after reconstitution. As shown in Figure 6A, chimeras reconstituted with WT BM cells exhibited normal pDC compartments (B220+120G8+ cells) in both the BM and spleen. In striking contrast, both single chimeras reconstituted with IkL/L BM cells and double chimeras exhibited the same block in IkL/L pDC differentiation at the B220–120G8+ stage in the BM, with few if any detectable IkL/L pDCs in the spleen. WT B6.Ly5SJL pDCs differentiated normally in the double chimeras reconstituted with WT and IkL/L BM cells, eliminating the possibility that IkL/L BM cells might exert a dominant-negative intercellular effect on pDC differentiation (not shown). These results demonstrate that the pDC block is due to a cell-intrinsic defect in IkL/L hematopoietic cells. Ikaros expression in pDCs To determine the relative expression of Ikaros and other family members during pDC maturation, we analyzed its expression, versus that of Aiolos and Helios, in pDCs and cDCs by RT-PCR. Ikaros was expressed in WT BM and splenic pDCs, and in splenic cDCs (Figure 6B), although at much lower levels than in thymocytes. Interestingly, Aiolos was expressed by all splenic DCs, but at barely detectable levels in the BM pDC population. Helios expression was not detected in any of the DC populations tested. These results suggest that Ikaros is the predominant family member expressed in BM pDCs.
Ikaros represses non-pDC gene expression
To gain insight into the mechanism of Ikaros function during pDC differentiation, we compared the gene expression profiles of WT and IkL/L BM CD11c+120G8+ pDCs. Three independent RNA samples were prepared for each genotype, and analyzed on pangenomic Affymetrix arrays. Most genes were similarly expressed between WT and IkL/L samples (note the global shape of the scatterplots in Figure 7A), including those previously found to be highly expressed in pDCs (ie, SpiB, Bcl-11a, and Siglec_H). These data provide further evidence that IkL/L BM pDCs belong to the pDC lineage. However, there were striking differences in the IkL/L transcriptome. Three-hundred and seventy genes were strongly overexpressed in IkL/L BM pDCs with 137 of these up-regulated more than 4-fold (Figure 7B; Table 2; Table S1). Furthermore, 52 of these genes were normally not expressed in WT BM pDCs (Figure 7C; Table 2 asterisks). In contrast, most of the 247 down-regulated genes showed only a modest decrease in expression (Figure 7B; Table S2), and only 4 of them were turned off in IkL/L BM pDCs. These experiments suggest that Ikaros deficiency results in a widespread and strong derepression of specific genes in immature pDCs. Interestingly, some of these deregulated transcripts are T- or B-cell specific. IkL/L BM pDCs strongly express Lck, pKC
Our data show that pDCs are selectively and severely reduced in mice with diminished Ikaros activity and that this phenotype is dose dependent on Ikaros. Loss of the pDC population, but not other DC subsets or hematopoietic lineages in IkL/L mice,35,36 allowed us to confirm the highly specific IFN -producing role of pDCs to influenza virus, and to TLR7 and TLR9 ligands. Moreover, we confirm that pDCs are essential in the early response to viral infection, as MCMV-infected IkL/L mice failed to produce high levels of IFN and control viral replication.
Although pDCs are absent in IkL/L mice, cDCs appear normal. Their numbers are slightly reduced in IkL/L collagenase-treated organs, but these cells respond normally when stimulated in vivo with MCMV or STAg, producing IL-12 and up-regulating maturation markers. Thus, the IkL/L mouse line provides a powerful tool to study pDC requirement in vivo, without previous external manipulation. Indeed, IkL/L mice have been valuable in revealing a second and novel wave of IFN
The lack of peripheral pDCs in IkL/L mice is likely due to a developmental block in the BM, where we have identified a putative pDC precursor population. These cells resemble WT BM pDCs in several aspects: (1) they express 120G8, Ly-6C, Flt-3, CD8 , and CD4, and exhibit a gene expression program similar to that of WT pDCs; (2) they present a typical plasmacytoid morphology; and (3) they produce IFN after stimulation with CpG oligonucleotides and, to a lesser extent, influenza virus. Since IkL/L BM pDCs express less TLR7 at the mRNA level (Table S2), and TLR7 mediates influenza-induced IFN production,59,60 our transcriptome data may explain why IkL/L pDCs respond less well to influenza stimulation. Moreover, IkL/L BM cells give rise to 120G8+ cells with similar kinetics as WT BM cells following Flt-3L stimulation in vitro, indicating that IkL/L progenitors can proceed through the initial steps of pDC differentiation. Interestingly, IkL/L BM 120G8+ cells are mostly Ly-49Q–, like the immature pDC subset recently identified in WT BM, which may represent the immediate precursors of the more mature Ly-49Q+ pDCs found in the BM and the periphery.54-56 It is therefore tempting to speculate that IkL/L pDCs are blocked at this immature Ly-49Q– stage of differentiation. However, IkL/L BM 120G8+ cells are also different in other respects—they lack B220, and express some markers of activation (CD8, MHC class II, CD40) but not others (CD80, CD86). Collectively, our data suggest that commitment to the pDC lineage occurs in the IkL/L BM, but that differentiation is blocked. Our results show that pDC-committed precursors in the BM show a cell-intrinsic requirement for Ikaros, as IkL/L BM pDCs do not terminally differentiate in vivo even if given a WT BM and stromal microenvironment, as shown in single and double chimera experiments. Although Flt-3/Flt-3L signaling has been shown to be essential for optimal pDC development22-24 and Flt-3 expression appears dependent on Ikaros,34 our studies suggest that this pathway is not defective in IkL/L BM pDCs, and notably, that Flt-3/Flt-3L signaling alone is not sufficient for pDC development. Indeed, IkL/L BM cells respond to Flt-3L stimulation in vitro but never give rise to mature cells. Moreover, cDC development and function appear normal in IkL/L mice, and their differentiation also depends on Flt-3/Flt-3L.61 How Ikaros regulates pDC differentiation may be linked to its proposed function as a transcriptional repressor. Our gene expression analyses show that a significant number of genes, not normally expressed in WT pDCs, are strongly up-regulated in IkL/L BM pDCs. These data suggest that Ikaros may act as a key repressor of non-pDC–specific genes during pDC differentiation, similar to Pax5 function in early B-cell development.62 Of importance, several T- and B-lineage–specific genes are highly expressed in IkL/L pDCs, suggesting that Ikaros is required to suppress lymphoid-related genes in pDCs. It is also noteworthy that the majority of deregulated genes code for adhesion molecules, cell surface receptors, extracellular matrix proteins, and secreted factors (Table 2; Figure S2), suggesting that an altered response to the BM environment may play an important role in the differentiation block seen here. Further functional investigation will be required to dissect the roles of these genes in pDC differentiation.
Our observation that most of the deregulated genes show increased mRNA expression supports the hypothesis that Ikaros acts mainly as a repressor, through its association with the NURD histone deacetylase complex or the CtBP and Sin3 corepressors. The fact that few transcriptional activators are strongly induced in IkL/L pDCs (Table 2) argues in favor of a direct influence by Ikaros on the affected genes. Indeed, 2 genes have previously been shown to be targets of Ikaros-mediated repression in other systems:
IkL/L mice carry a
Our results, together with previous work highlighting the role of Ikaros in hematopoiesis and DC differentiation,30,44 should provide insights into the molecular events required for the development of each DC lineage. Previous data illustrate that mice expressing a dominant-negative Ikaros mutation exhibit a complete block in the development of all DCs, and animals bearing a null mutation produce some CD8 In conclusion, we have shown that our mouse model bearing a hypomorphic mutation for Ikaros specifically lacks plasmacytoid dendritic cells, but not conventional dendritic cells, in the periphery. We have demonstrated that high levels of Ikaros are not necessary for engagement of BM precursors into the pDC lineage but are required for terminal differentiation of BM pDCs. This block is linked to a failure of IkL/L pDCs to silence the expression of many genes, some of which encode proteins important in intercellular crosstalk. Further studies are now required to determine the specific contribution of these genes to pDC differentiation.
Contribution: D.A., M.D., C.A.-P., T.D., S.H.R., P.K., and S.C. designed and performed experiments; G.T. and C.A.B provided valuable reagents and intellectual input; and M.D., P.K., and S.C. wrote and edited the manuscript. Conflict-of-interest disclosure: The authors declare no competing financial interests. D.A., M.D., P.K., and S.C. contributed equally to this work.
We thank G. Yap (Brown University) for the kind gift of STAg; M. Sellars for critical reading of the paper; S. Duhautbois-Boine, G. Kimmich, and G. Brizard for help; C. Thibault, C. Grussenmeyer, and D. Dembélé for microarray experiments and analysis; J. Barths, C. Ebel, and I. Durand for flow cytometry; and F. Memedov and M. Gendron for animal husbandry. This work was supported by institute funds from INSERM, CNRS, and the Hôpital Universitaire de Strasbourg; a grant from the Association pour la Recherche sur le Cancer (ARC); and the Centre National de Ressources en Génomique (Programme Affymetrix) (P.K. and S.C.). M.D. was supported by an ATIPE (Action Thématique et Incitative sur Programme) grant from the CNRS and a grant from the ARC. S.H.R. was supported by the CNRS, the Fondation pour la Recherche Médicale, and the Philippe Foundation. D.A. was supported by National Institutes of Health (NIH) grants AI52861 and AI58066, and is the recipient of a Career Development Program Award from the Leukemia and Lymphoma Society. C.A.B. was supported by NIH grant AI55677.
Submitted March 6, 2006; accepted July 27, 2006.
Prepublished online as Blood First Edition Paper, August 15, 2006; DOI 10.1182/blood-2006-03-007757.
The online version of this article contains a data supplement.
An Inside Blood analysis of this article appears at the front of this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
Correspondence: Philippe Kastner and Susan Chan, IGBMC, BP 10142, 67404 Illkirch Cedex, France; e-mail: scpk{at}igbmc.u-strasbg.fr.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
was measured using the IFN
,pT
5 and VpreB1, which encode the surrogate light chains of the pre-BCR. Also intriguing is the strong induction of CD56 (NCAM1, 
correspond to the estimated number of nonspecific changes ("Materials and methods"). (C) Number of up-regulated genes in IkL/L samples whose expression was not detected in WT samples (
) or were expressed in WT but not detected in IkL/L samples (
).
.