extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

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Extracellular ATP and P2X7 receptor exert context-specific immunogenic effects after immunogenic cancer cell death

Extracellular ATP and P 2 X 7 receptor exert context-specific immunogenic effects after immunogenic cancer cell death

Immunogenic cell death (ICD) facilitates danger signallingdriven trafficking of damage-associated molecular patterns (DAMPs) like extracellular ATP (eATP). 1,2 The binding of eATP to P 2 X 7 receptor triggers immunogenic signalling, 3 which (along with other factors) converts the dying cancer cells into an effective anticancer vaccine. 3 Endoplasmic reticulum (ER) stress is central to ICD, 1 on the basis of which ICD inducers are subdivided into two types, 1 that is, Type I (e.g., some chemotherapies), which elicit danger signalling through 'collateral' non-lethal ER stress, 1 and Type II (e.g., hypericin-photodynamic therapy (Hyp-PDT)), which elicit danger signalling via 'focused' lethal ER stress. 1,4 Type II and Type I ICD inducers differ on several levels, for example, plasticity of danger signalling and the trafficking mechanisms of DAMPs. 4 In fact, eATP was found to be absent during Newcastle disease virus (NDV)-induced Type II ICD despite the induction of macroautophagy (a Type I ICD-associated, eATP-trafficking mechanism). 2,5 Moreover, we have established that Hyp-PDT-induced eATP is PERK and secretory pathwaydependent, 6 while being independent of macroautophagy 7 or chaperone-mediated autophagy. 8 This raised an important questionlike in the case of NDV-induced ICD, could eATP be dispensable or a partial immunogenic signal for Hyp-PDTinduced ICD?
To this end, we decided to gain further insights into the eATP-trafficking mechanism and its immunogenic potential following Hyp-PDT. To address the contribution of the pannexin/connexin-caspase axes 2 that elicits eATP secretion (in response to Type I ICD inducers but remains enigmatic in the Type II settings), we utilized the pan-pannexin/ connexin inhibitor, carbenoxolone (CBX). In CT26 cells treated with Hyp-PDT, CBX pretreatment failed to reduce eATP (Figure 1a), thereby suggesting the dispensability of pannexins/connexins. Next, we addressed the role of caspase activity using the pan-inhibitor, zVAD-fmk. Interestingly, zVAD-fmk significantly reduced Hyp-PDT-induced eATP ( Figure 1a). Considering the previously demonstrated role of casp-8 in ICD 1,6 we wondered whether this caspase was mediating eATP secretion. Interestingly, CT26 cells expressing caspase-8 shRNA (casp-8 shRNA) also exhibited significantly reduced eATP following Hyp-PDT ( Figure 1a).
The regulation of eATP secretion by casp-8 was unexpected, as our previous study found casp-8 to be dispensable for Hyp-PDT-induced ICD, in vivo. 6 This suggested that eATP secretion may not be crucial for Hyp-PDTinduced ICD, in vivo. To resolve this, we utilized the CT26-BALB/c mice prophylactic vaccination model. Immunogenic effects of eATP were blocked using either Apyrase or Apy (an ATP-degrading enzyme, Figure 1b) or a 2,3-dialdehyde derivative of ATP, that is, oxidized-ATP (Oxi-ATP, a P 2 X 7 receptor antagonist) or a combination of both (i.e., Apy+Oxi-ATP). 3 Approximately 70% of the mice immunized with Hyp-PDT-based vaccine efficiently rejected the formation of CT26 tumours at the challenge site ( Figure 1c). Interestingly, eATP degradation or blockade of P 2 X 7 receptor, alone, failed to strongly reduce the tumour-rejecting immunity (Figure 1c). On the other hand, only the combination of Apy+Oxi-ATP significantly reduced the vaccine's tumourrejecting capacity (Figure 1c). Thus, eATP, despite being ubiquitously secreted after Hyp-PDT, 6,7,8 only acts as a partial immunogenic signal, and thus singular blockade of either eATP or its P 2 X 7 receptor is unable to reduce the immunogenic potential of the vaccine.
These results are unprecedented because eATP and P 2 X 7 receptor had been shown to act in a synergistic manner. 1,2,3 Here, we rather observed a potentiating effect, that is, blockade of either eATP or P 2 X 7 receptor did not, but combined blockade significantly reduced ICD's immunogenic potential. Thus, our results suggest that the mere presence of eATP does not ensure the presence of corresponding immunogenic activity in all contexts. Moreover, a certain degree of redundancy exists on the level of purinergic receptor agonists, and thus these results may also point to the release of such (as-yet-uncharacterized) agonists from dying cells. Lastly, these observations are based on the heterotopic (subcutaneous) tumour model; it would be crucial to reanalyze the role of eATP in an orthotopic tumour model to overcome immunological variations stemming from incompatibility between the transplanted cancer type and the surrounding tissue.

Figure 1
Extracellular ATP and P 2 X 7 receptor together potentiate ICD in cancer. (a) CT26 cells were treated with Hyp-PDT (dosage: 150 nM Hypericin preincubation for 16 h followed by light irradiation with a total fluence of 2.70 J/cm 2 ) as described previously 6 and recovered for eATP analysis 1 h post treatment. Depending on the settings (as indicated in the legends above the graphs), the cells were preincubated with CBX (100 μM for 1 h) or zVAD-fmk (25 μM for 30 min). Alternatively, CT26 cells expressing control shRNA (CO-shRNA) or casp-8 shRNA were utilized as described previously. 6 Extracellular ATP was detected using the standard luciferin-luciferase bioluminescence assay. 7 Here, n = 3-4, mean ± S.E.M., Student's t-test, **Po0.01 and ***Po0.001, NS, non-significant; CNTR, untreated controls. (b) In another case, CT26 cells were treated with Hyp-PDT as described above and incubated for 15 min post recovery with Apyrase (Apy; 10 U/ml); eATP was then analyzed as described above. (c) For testing of immunogenicity, the CT26-BALB/c mice model was utilized. 6 Here, the CT26 cells were treated with Hyp-PDT followed by 'vaccine' preparation as described previously. 6 In certain cases, the vaccines were mixed/co-injected with either Apy (10 U/ml for 15 min) or Oxi-ATP (4 mg/kg per mouse) or both (Apy+Oxi-ATP). These respective vaccines were given twice with an interval of 7-8 days between vaccinations in one of the flanks of the syngenic BALB/c mice. About 8-10 days following the vaccination regimen, the vaccinated mice were challenged on the contra-lateral flank with live CT26 cells. Thereafter, the mice were monitored for the occurrence of CT26 tumours at the challenge site. Here, n = 10 for PBS, n = 12 for Hyp-PDT, n = 12 for Hyp-PDT+Apy, n = 12 for Hyp-PDT+Oxi-ATP and n = 6 for Hyp-PDT+Apy+Oxi-ATP, Fisher's exact test; *Po0.05, **Po0.01 and ***Po0.001; NS, non-significant


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Evolutionarily conserved autoregulation of alternative pre-mRNA splicing by ribosomal protein L10a

Alternative splicing of pre-mRNAs can regulate expression of protein-coding genes by generating unproductive mRNAs rapidly degraded by nonsense-mediated mRNA decay (NMD). Many of the genes directly regulated by alternative splicing coupled with NMD (AS-NMD) are related to RNA metabolism, but the repertoire of genes regulated by AS-NMD in vivo is to be determined. Here, we analyzed transcriptome data of wild-type and NMD-defective mutant strains of the nematode worm Caenorhabditis elegans and demonstrate that eight of the 82 cytoplasmic ribosomal protein (rp) genes generate unproductively spliced mRNAs. Knockdown of any of the eight rp genes exerted a dynamic and compensatory effect on alternative splicing of its own transcript and inverse effects on that of the other rp genes. A large subunit protein L10a, termed RPL-1 in nematodes, directly and specifically binds to an evolutionarily conserved 39-nt stretch termed L10ARE between the two alternative 5′ splice sites in its own pre-mRNA to switch the splice site choice. Furthermore, L10ARE-mediated splicing autoregulation of the L10a-coding gene is conserved in vertebrates. These results indicate that L10a is an evolutionarily conserved splicing regulator and that homeostasis of a subset of the rp genes are regulated at the level of pre-mRNA splicing in vivo.

INTRODUCTION
Alternative splicing of precursor messenger RNAs (pre-mRNAs) expands proteome diversity in multicellular organisms (1). Indeed, recent genome-wide transcriptome analyses revealed that more than 90% of protein-coding genes in humans produce multiple mRNA isoforms (2,3). On the other hand, early bioinformatics analysis of ex-pressed sequence tags (ESTs) and cDNA sequence data predicted that a significant fraction of the alternatively spiced mRNA isoforms have premature termination codons (PTCs) within open reading frames (ORFs) (4)(5)(6). Recently, alternative splicing coupled with NMD (AS-NMD), also referred to as regulated unproductive splicing and translation (RUST), has been shown to be one of the mechanisms for regulating gene expression levels by knocking down or knocking out essential factors for NMD (7,8).
Many of well-characterized examples of genes directly regulated by AS-NMD are related to RNA metabolism (9)(10)(11). In a number of cases, they are splicing factors and autoregulate their own transcripts to maintain their own abundance in a negative feedback loop. Such examples include general splicing factors such as SR and SR-like proteins (12)(13)(14)(15)(16) and heterogeneous nuclear ribonucleoproteins (hnRNPs) (17)(18)(19)(20), tissue-specific splicing factors (21)(22)(23) as well as core spliceosomal proteins (24,25). Genomewide search for AS-NMD target mRNAs in Drosophila S2 cells by utilizing a custom splicing-sensitive microarray also identified numerous genes related to RNA metabolism such as splicing and translation (26).
In C. elegans, critical NMD factors encoded by a series of the smg genes are dispensable for viability and fertility (27) unlike in vertebrates (28)(29)(30). Therefore, the smg mutants have been utilized for characterization of unproductive splice variants or other natural NMD targets in vivo (31)(32)(33)(34)(35)(36). Splicing-sensitive microarray analysis of 352 known cassette exons in embryonic total RNAs revealed that 30 of them, including 10 in splicing factor genes, produce PTCcontaining mRNA isoforms upon alternative splicing, likely representing direct targets for AS-NMD (37). Global analyses of total RNAs from L3 or older worms by genome-scale tiling arrays and massively parallel sequencing revealed that ∼20% of genes likely produce NMD target transcripts and that splicing factor genes are enriched in those that produce PTC-containing NMD targets (38). These large-scale analyses, however, suggested that a large fraction of the natural NMD targets are destabilized independently from PTCs generated upon alternative splicing (37,38) and are related to the presence of upstream ORF (uORFs) or longer 3 UTRs (38).
Here, we performed mRNAseq analysis of a wild-type strain N2 and the smg-2 (yb979) mutant (39) to comprehensively search for NMD target mRNAs generated by alternative splicing in C. elegans. In this study, we focus on ribosomal protein (rp) genes because we confirmed expression of novel and unproductively spliced mRNAs from four rp genes in the smg-2 mutant in addition to four rp genes already known to produce unproductive splice variants in an early study (33). We elucidate homeostatic regulation of all these rp genes at the alternative splicing level. We also demonstrate that ribosomal protein L10a, like splicing factors, regulates alternative splicing of its own pre-mRNA in a negative feedback loop by directly binding to an evolutionarily conserved element.

Worm culture and microscopy
Worms were cultured following standard methods. The strains used are N2 and KH1668: smg-2 (yb979) I. Transgenic worms were generated as described previously (40). Images of fluorescence reporter worms were captured using a fluorescence compound microscope (DM6000B, Leica) equipped with a color, cooled CCD camera (DFC310FX, Leica) and processed with Photoshop (Adobe).

RNA preparation, mRNA-seq and RT-PCR
Total RNAs from synchronized worms were extracted as described previously (40,41). mRNA-seq was performed as described previously (41). The sequence data for N2 and the smg-2 mutant are available in Sequence Read Archive (SRA) under accession numbers DRA000864 (41) and DRA002612 (this study). The numbers of uniquely mapped 35-nt sequence tags for N2 and KH1668: smg-2 (yb979) deposited in DRA002612 were 6 583 548 and 6 964 488, respectively. At least one read from either of the strains was mapped to 15 479 (76.3%) of the 20 290 genes in WormBase and 1383 genes (6.8%) were predicted to contain differentially expressed exons by the method described in (41). Total RNAs from cultured mammalian cells were extracted by utilizing Sepasol RNA I Super (nacalai tesque) and RNeasy Mini (QIAGEN). Total RNAs from transfected cells were and treated with RQ1 DNase (Promega) and DNase (QIAGEN). RT-PCR was performed essentially as described previously (40,41). Sequences of the primers used in the RT-PCR experiments are available in Supplementary Table S1. RT-PCR products were analyzed by using BioAnalyzer (Agilent). Sequences of the RT-PCR products were confirmed by direct sequencing or by cloning and sequencing. Sequences of the novel and unproductive mR-NAs were deposited in GenBank/EMBL/DDBJ databases under accession numbers LC006013, LC006014, LC006015 and LC006016 for C. elegans rpl-26, rpl-30, rps-22 and ubl-1, respectively, and LC010245 and LC010246 for human RPL10A.

RNAi
RNAi experiments by feeding were performed as described previously (42) by utilizing RNAi feeding library (Open Biosystems; Geneservice) for rpl -1, rpl-3, rpl-12, rpl-26, rpl-30, rps-22 and ubl-1. For knocking down the rpl-7A gene, we constructed pL4440-RPL-7A by cloning a genomic DNA fragment spanning from rpl-7A exon 4 to exon 5 at the Eco RV site of pL4440 vector (from A. Fire) by using In-Fusion HD Cloning Kit (Clontech). Escherichia coli strain HT115 (DE3) was transformed with pL4440-RPL-7A and used for RNAi by feeding. Sequences of the primers are available in Supplementary Table S2.
RNAi experiments by soaking were performed essentially as described previously (43,44). The RNAs were synthesized by in vitro transcription with T7 (Takara) or SP6 (Promega) RNA polymerase. Sequences of the primers used in amplifying cDNA fragments as templates for the in vitro transcription are available in Supplementary Table S3.

Construction of expression vectors for C. elegans genes
Fluorescence rpl-1 splicing reporter minigenes were constructed essentially as described previously (40,45). Briefly, the rpl-1 genomic DNA fragment was cloned into Gateway pENTR-L1/R5 vector (Invitrogen) by utilizing BP Clonase II Plus (Invitrogen). Mutagenesis and deletion of L10ARE were performed by utilizing Quickchange II (Stratagene). Expression vectors were constructed by homologous recombination between the genomic DNA cassette, a fluorescent protein cassette in pENTR-L5/L2 vector and a destination vector pDEST-eft-3p or pDEST-myo-3p (46) by utilizing LR Clonase II Plus (Invitrogen). Sequences of the primers used in the plasmid construction are available in Supplementary Table S2.
Expression vectors of RPL-1 and RPL-30 were also constructed by utilizing Gateway system (Invitrogen). RPL-1 and RPL-30 cDNAs were amplified and cloned in pENTR/D-TOPO vector (Invitrogen). The expression vectors were constructed by homologous recombination between the Entry vectors and pDEST-eft-3p or pDEST-Cold-ZZ (sequence information is available upon request to H.K.) with LR Clonase II (Invitrogen). All constructs were confirmed by sequence analysis. Sequences of the primers Nucleic Acids Research, 2016, Vol. 44, No. 12 5587 used in the construction are available in Supplementary Table S2.

UV crosslinking and electrophoretic mobility shift assay (EMSA)
Recombinant full-length RPL-1 and RPL-30 proteins were expressed as His-ZZ-fused proteins by cold-induction (47) in E. coli strain BL21(DE3)pLysS, purified by using Ni-NTA agarose (QIAGEN), quantified by using Micro BCA Protein Assay Kit (PIERCE) and analyzed by CBB staining of neutral PAGE gel (Invitrogen). 32 P-labeled RNA probes were prepared as described previously (48). The templates for the in vitro transcription were amplified from genomic DNA or the reporter minigenes by PCR. The sequences of the primers used are available in Supplementary Table S3. In vitro binding reactions were performed in the presence of 100 ng/l E. coli tRNAs and 50 ng/l bovine serum albumin in 25 l of RNA binding buffer (150 mM KCl in 20 mM HEPES-KOH (pH 7.9) with 5% glycerol, 1% Triton X-100, 1 mM DTT and 0.1 mM PMSF) for 30 min at 20 • C. UV crosslinking (46) and EMSA (48) were performed essentially as described previously except that the denatured proteins were separated by using NuPAGE gel (Invitrogen).

Construction of expression vectors for mammalian genes
The human RPL10A WT reporter cassette was constructed by cloning RPL10A genomic DNA fragments spanning from exon 3 to exon 5 into pENTR/D-TOPO vector (Invitrogen). L10ARE was deleted by utilizing Quickchange II (Stratagene) for the RPL10A DEL reporter cassette. The reporter minigenes were constructed by homologous recombination between the Entry vectors and pDEST-cDNA3 (45) by utilizing LR Clonase II (Invitrogen). Sequences of the primers used in the construction are available in Supplementary Table S2.
Entry vectors for human L10a and L26 were constructed by cloning cDNA fragments into pENTR/D-TOPO vector (Invitrogen). The Expression vectors were constructed by homologous recombination between the Entry vectors and pDEST-ME18S (H.K.) by utilizing LR Clonase II (Invitrogen). All constructs were confirmed by sequence analysis. Sequences of the primers used in the construction are available in Supplementary Table S2.

Cell culture
HeLa, HEK293T and NIH3T3 cells were cultured in DMEM (nacalai tesque) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin mixed solution (nacalai tesque). HeLa cells were grown to 90% confluence and treated with 100 g/ml emetin (SIGMA) for 6 h. The human RPL10A reporter minigenes and the expression vectors were co-transfected at the ratio of 1:3 into NIH3T3 cells by utilizing FuGENE HD (Roche).

Eight rp genes produce unproductively spliced mRNAs in C. elegans
To identify unproductive splice variants stabilized in the NMD-defective mutant, we compared mRNA-seq data to search for genes whose mRNAs appeared to be differentially spliced between the wild-type strain N2 and the smg-2 (yb979) mutant. For this purpose, we sequenced poly(A) + RNAs from synchronized L1 larvae because we previously noticed enrichment of neuron-specific minor variants at this stage (45) and successfully identified 24 alternative splicing events regulated by a neuron-specific splicing factor UNC-75 in the same way (41). We estimated significance of difference in inclusion levels between N2 and smg-2 for each exon in all RefSeq gene models (41), and noticed enrichment of the rp gene exons in the candidates (11 out of top 50). We therefore inspected distribution of the sequence reads for each of all the 82 cytoplasmic ribosomal protein genes in C. elegans (Ribosomal Protein Gene Database (RPG), http: //ribosome.med.miyazaki-u.ac.jp/), and analyzed splicing patterns of candidate transcripts in N2 and the smg-2 mutant by reverse transcription-polymerase chain reaction (RT-PCR). Sequencing of the RT-PCR products revealed that rpl-26 and rpl-30, encoding 60S subunit proteins L26 and L30, respectively, and rps-22 and ubl-1, encoding 40S subunit proteins S22 and S27a, respectively, have smg-2specific mRNA isoforms ( Figure 1) in addition to the four rp genes known to express unproductive mRNAs (33). The novel smg-2-specific mRNA isoforms contain PTCs ( Figure  1), consistent with the idea that they are unproductive mR-NAs and are rapidly degraded in the wild-type by NMD.

Homeostatic regulation of the rp genes by alternative pre-mRNA splicing
As ribosomal proteins are principally RNA-binding proteins present in the nucleus and many splicing regulators autoregulate production of their own unproductive mRNA isoforms, we tested possibilities that the eight ribosomal proteins are involved in the splicing regulation of their own pre-mRNAs. To systematically knockdown each of the rp genes, we fed synchronized L4 larvae of the smg-2 mutant with bacteria expressing double-stranded RNA targeting the rp gene and cultured the worms for another 6, 12, 24 or 36 h. In the control experiments with bacteria carrying an empty vector, total amounts of the two isoforms and as well as ratios of the productive isoforms to the total remained rather constant ( Figure 2A, left panels, Supplementary Figure S1 and Figure 2B, blue lines) regardless of substantial growth of the worms during this period. When rpl-1 is knocked down, the total amount of the rpl-1 mRNAs gradually decreased along with time ( Figure 2A, top right panel), confirming that rpl-1 was effectively knocked down. Noticeably, the ratio of the productive rpl-1 mRNA to the total gradually increased ( Figure 2A, top right panel and Figure 2B, top left panel), likely compensating for the reduction in the total amount. In contrast, the ratios of the productive mRNA isoforms gradually decreased for rpl-7A (  els). Analogous results were obtained when any one of the other seven rp genes were knocked down: increase in the ratio of the productive mRNA isoforms for the targeted gene and decrease for the rest (Supplementary Figure S1 and Figure 2B). These results indicated that expression of these rp genes are regulated by their own products either directly or indirectly at the level of alternative splicing, which contributes to the homeostasis of the rp gene products.

A 40-nt stretch is highly conserved in orthologous introns of genes encoding ribosomal protein L10a in metazoans
It has been reported that nucleotide sequences of the alternatively spliced introns of the rpl-1, rpl-3, rpl-7a and rpl-12 genes are exceptionally conserved (66-93% identity) among related nematode species C. briggsae and C. remanei compared to those of constitutive introns (33). We compared genomic sequences of the rpl-26, rpl-30, rps-22 and ubl-1 genes between C. elegans and related nematodes and found that the alternative introns of these genes are also highly conserved (68-84% identity) except for C. japonica rps-22 (53%) (Supplementary Figure S2), suggesting that alternative splicing regulation is also conserved for these genes.
During the course of nucleotide sequence comparison, we found that a 39-nt stretch residing between the two alternative 5 splice sites of rpl-1 intron 1 in C. elegans is conserved in orthologous introns of orthologous genes encoding ribosomal protein L10a in vertebrates and a fly ( Figure 3). Here, the orthologous introns are defined as those that reside at exactly the same position in the coding sequences for the evolutionarily conserved ribosomal proteins. In contrast, such an evolutionarily conserved stretch was not found for rpl-3 intron 1, although analogous alternative splicing regulation of the orthologous introns has been reported for the human RPL3 gene (49) and predicted for the Drosophila RpL3 gene (FlyBase, http://flybase.org). We therefore focused on the rpl-1 gene in elucidating mechanisms for autoregulation of alternative pre-mRNA splicing in the following sections.

Fluorescence rpl-1 splicing reporters for visualizing alternative splicing regulation in vivo
In order to visualize the splicing patterns of the rpl-1 mR-NAs in vivo, we constructed a pair of wild-type (WT) rpl-1 fluorescence reporter minigenes that contain a genomic fragment spanning from exon 1 through exon 2 ( Figure  4A). The minigenes were designed so that expression of red (RFP) and green (GFP) fluorescent proteins indicated the use of the upstream (or productive) and the downstream (or unproductive) splice sites, respectively ( Figure 4A). When the rpl-1 WT reporter minigenes were expressed under the control of a ubiquitous promoter, RFP was predominantly expressed and GFP was modestly expressed without apparent tissue-specificity ( Figure 4B). Since the reporter worms were fairly sick, we utilized a body wall muscle-specific promoter to drive expression of the reporter (Supplementary Figures S3 and S4A) in the following experiments.
To test splicing regulation of the rpl-1 WT reporter, we knocked down rpl-1, rpl-26 or rpl-30 in the reporter worms. As expected, the productive splice site was predominantly selected upon knockdown of the endogenous rpl-1 gene, whereas the unproductive splice site was preferred upon knockdown of rpl-26 or rpl-30 ( Figure 4C). Expression of the fluorescent proteins was consistent with the splicing change of the reporter mRNAs (Supplementary Figure  S4). Next, we tested the effects of RPL-1 overexpression on the rpl-1 WT reporter. In contrast to the knockdown experiment, transgenic expression of RPL-1 dramatically increased expression of GFP ( Figure 4D), while expression of RPL-30 had little effect ( Figure 4E). These results indicated that the fluorescence rpl-1 WT reporter specifically reflected the negative feedback regulation of the endogenous rpl-1 gene.

L10ARE is essential for the splicing regulation of the rpl-1 splicing reporter in vivo
To test the involvement of the evolutionarily conserved 39nt stretch between the two alternative splice sites in the regulation of the rpl-1 splicing reporter, we constructed a pair of deletion mutant (DEL) minigenes in which the entire stretch was deleted ( Figure 4A). The rpl-1 DEL reporter worms also expressed both of RFP and GFP (Supplementary Figures S3 and S5A) but were no longer affected by knockdown of rpl-1, rpl-26 or rpl-30 ( Figure 4F and Supplementary Figure S5) or by overexpression of RPL-1 ( Figure  4G). These results indicated that the conserved stretch is an essential cis-element for the splicing regulation of the rpl-1 reporter. We therefore designated the stretch as ribosomal protein L10a regulatory element (L10ARE).

RPL-1 protein directly and specifically recognizes L10ARE in vitro
To test whether RPL-1 protein can directly and specifically recognize rpl-1 pre-mRNA, we performed in vitro binding

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C. elegans

human T C -C C T C T G A --G G T G C G T G G C G T C C A T G G C ---A C ---A C C G G G T A A T T C G -G G C T C T ---G T C -C G C A G chicken T T -T T T T T A C --T G T G T A A A A A A T G A G A A T C T A G T C C T T T C T G A --A A T G C A -T T T T T C T A C A T T -C A C A G
X. tropicalis  Figure S6B). RPL-1 strongly bound to probe 3, moderately to probe 1 and weakly to probes 2 and 4 in UV crosslinking experiments, whereas RPL-30 was only faintly crosslinked with these probes (Figure 5B, top panel). Consistent results were obtained in EMSAs (Supplementary Figure S6C). These results indicated that RPL-1 can directly bind to rpl-1 pre-mRNA in a sequence-dependent manner.

D. melenogaster
As probe 3 was the only fragment that contains the L10ARE stretch, we next asked whether L10ARE is required for RPL-1 to specifically recognize the rpl-1 pre-mRNA by using probes 5 and 6 that have sequences derived from the WT and the DEL reporter minigenes, respectively ( Figure 5A and Supplementary Figure S6A). RPL-1 was strongly crosslinked with probe 5 and weakly to probe 6, whereas RPL-30 was only weakly crosslinked with probe 5 ( Figure 5B, bottom panel). Consistent results were obtained in the EMSAs (Supplementary Figure S6C). These results indicated that the RPL-1 protein directly and specifically binds to the rpl-1 pre-mRNA via L10ARE in vitro.

Autoregulation of alternative pre-mRNA splicing of the RPL10A gene is conserved in mammals
To ask whether L10ARE-mediated splicing autoregulation by L10a is evolutionarily conserved in metazoans, we first tried to detect a novel and unproductive splice variant mRNA from the human RPL10A gene. The putative L10ARE stretch resides in intron 3 and the unproductive variant was expected to have a PTC and be degraded by NMD. We therefore treated HeLa cells with emetine, an inhibitor of protein biosynthesis, to stabilize NMD target mRNAs (50), which was confirmed by stabilization of an unproductively spliced RPL3 mRNA (49) (Figure 6A, bottom panel). In addition to the productive RPL10A mRNA, we discovered two novel and emetine-stabilized RPL10A mRNA isoforms: one utilizing a novel 5 splice site downstream of the putative L10ARE stretch and one with intron 3 retention ( Figure 6A, top panel and Supplementary Figure S7). These results indicated that the alternative splice donor site does exist in intron 3 of the human RPL10A gene (Figure 3) for the unproductive mRNA rapidly degraded by NMD.
To analyze splicing regulation of the human RPL10A gene, we constructed a wild-type (WT) RPL10A reporter minigene that contains a genomic fragment spanning from exon 3 through exon 5 ( Figure 6B). When the minigene was expressed in NIH3T3 cells, either of the two alterna-   tive splice donor sites were utilized ( Figure 6C, lanes 1-3). When co-expressed with L10a, the upstream (or productive) splice site was less efficiently selected (lane 2) compared to co-expression with RFP (lane 1) or L26 (lane 3). These results indicated that L10a specifically switches the donor site for RPL10A intron 3 from the upstream to the downstream sites in a similar way to C. elegans rpl-1 autoregulation.
To test whether the autoregulation of the RPL10A gene is dependent on the putative L10ARE stretch, we constructed a deletion mutant (DEL) reporter minigene in which the stretch was entirely deleted ( Figure 6B). The splicing pattern of the DEL reporter transcript was less significantly affected by L10a overexpression ( Figure 6C, lanes 4-6), confirming that L10a autoregulates alternative splicing of the RPL10A pre-mRNA primarily via L10ARE in mammals like in C. elegans.

DISCUSSION
In the present study, we analyzed the mRNA-seq data from the WT and the NMD-deficient smg-2 mutant strains of C. elegans to search all the 82 cytoplasmic rp genes for AS-NMD target mRNAs and identified eight rp genes, including four known genes (33), as regulated by AS-NMD. In the early study, Mitrovich and Anderson demonstrated that transgenic expression of RPL-12 protein affected alternative splicing of the endogenous rpl-12 pre-mRNA in C. ele- gans (33), suggesting autoregulation of splice site selection by a ribosomal protein in metazoans. We demonstrated here by knocking down the endogenous rp genes that alternative pre-mRNA splicing of not only rpl-12 but all the eight rp genes with the AS-NMD isoforms are actively regulated for homeostatic control of their expression levels. We also demonstrated that RPL-1 can directly bind to its own pre-mRNA via the evolutionarily conserved element L10ARE to switch the 5 splice sites of intron 1 for negative feedback regulation of its own expression like splicing regulators ( Figure 7).
AS-NMD has been reported for some of the rp genes in other organisms. Global search for AS-NMD target mR-NAs in Drosophila melanogaster S2 cells by utilizing a custom splicing-sensitive microarray identified five rp genes as regulated by AS-NMD (26). Among them, only RpL10Ab, encoding L10a, is overlapping with those in C. elegans as reported here. In human, AS-NMD mRNAs have been reported for RPL3 and RPL12 (49) besides RPL10A demonstrated here. These three genes were characterized after C. elegans orthologues and therefore global search for AS-NMD target rp genes in mammals are yet to be done. In Saccharomyces cerevisiae, most of protein-coding genes lack introns yet the rp genes are unusual in that they are over-represented among intron-containing genes (51,52). The introns in the RPS21B, RPS14B, RPS9A and RPS9B genes function to repress rather than to increase steadystate mRNA levels (51), suggesting regulatory roles for these introns. Thus, a limited subset of the rp genes are regulated at the splicing level in each organism. The genes encoding L10a are unique among the rp genes in that they commonly express AS-NMD target mRNAs in metazoans. Orthologues of L10a in Bacteria and Archaea, termed L1, have also been reported to autoregulate their own expression by directly binding to their own polycistronic mRNAs to repress translation (53,54), suggesting the physiological significance of fine-tuning gene expression of this evolutionarily conserved protein. L10a is modeled as a part of highly flexible L1 stalk comprised of helices 76, 77 and 78 of 28S rRNA in eukaryotic ribosomes (55) after crystal structure of prokaryotic ribosomes (56). The nucleotide sequence or predicted secondary structure of L10ARE, however, is not related to those of the L1 stalk. As the mode of target RNA recognition by L1 is considered to be highly conserved during evolution (54), other element(s) outside of L10ARE may also be involved in the specific recognition of its own pre-mRNA by L10a. Consistent with this idea, the entire nucleotide sequences between the two 5 -splices sites are highly conserved within the genus Caenorhabditis (33).
We demonstrated an extraribosomal function of L10a as a sequence-specific splicing regulator. Similar extraribosomal and autoregulatory functions have been described in the literature for some of the ribosomal proteins in eukaryotes, most of which rely on their properties as sequenceand structure-specific RNA-binding proteins. S. cerevisiae L30 (57) and human S13 (58) have been demonstrated to bind to their own pre-mRNAs to repress excision of their introns. Yeast L30 also represses translation of its own mature mRNA (59). Xenopus laevis L4 somehow represses splicing its own pre-mRNA to promote endonucleolytic cleavage (60). What is unique to L10a, however, is that it can alter splice site choice, although molecular mechanisms for splicing autoregulation by human RPL3 have not yet been fully elucidated (49,61,62). Considering their ubiquitous expression as house-keeping genes and high conservation of their amino acid sequences, it is reasonable to suggest that ribosomal proteins can potentially regulate homeostasis of gene expression as sequence-and structure-specific RNAbinding proteins. As L10ARE is 45 nucleotides apart from the upstream 5 -splice site in C. elegans and further apart in other species shown in Figure 3, repression of the upstream sites may not be merely due to steric hindrance of spliceosome assembly. L10ARE is required for the specific binding of L10a in vitro ( Figure 5 and Supplementary Figure S6) and splicing regulation by L10a in vivo (Figures 4 and 6), but not sufficient to switch splice site choice in a heterologous context (data not shown), suggesting that other elements as well as trans-acting factors may be involved in the splicing autoregulation by L10a.
We revealed unexpected crossregulation of the AS-NMD events between the eight rp genes in C. elegans. Crossregulation between paralogous genes via direct and specific binding has been reported for ribosomal proteins yeast S9 (51), S14 (63) and mouse L22/L22-like1 (64) as well as for a family of splicing regulators (65), which may be explained by duplication of genes that had already acquired autoregulation mechanisms. The crossregulation of the AS-NMD events between the rp genes is reminiscent of yet in good contrast to a recent report that RBFOX2 crossregulates AS-NMD of other splicing regulator genes in mouse embryonic stem cells (66). In the case of RBFOX2, it controls autoregulation of other splicing regulators by directly binding to canonical UGCAUG motifs in target pre-mRNAs (66). In the case of the C. elegans rp genes, on the other hand, it appears that not a single gene is dominant to the others but all are equivalent and mutually dependent. A likely mechanism, assuming that each of the ribosomal proteins can somehow autoregulate AS-NMD of its own transcript, is that the apparent crossregulations are all indirect secondary effects caused by excess amounts of free ribosomal proteins upon knockdown of one of the rp genes. This idea is supported by the results demonstrating that the ratios of the productive mRNAs more prominently decreased upon lack of proteins in the same subunit than lack of those in the other subunit ( Figure 2B).
As the ribosomes are abundant in cells and are assembled through a complex series of highly regulated steps (67,68), it is generally considered that the cells need to produce equimolar amounts of ribosomal proteins. Indeed, recent bioinformatics analysis of mRNA-seq data from various cell lines and tissues indicated that the molar ratio of mR-NAs varies less than 3-fold for most of the human rp genes, with little tissue specificity (69). Heterozygous mutations in many of the rp genes have been reported to cause remarkable common phenotypes in metazoans presumably due to haploinsufficiency. In human, haploinsufficiency in any of 10 rp genes are considered to be the cause of Diamond-Blackfan anemia (DBA) (70). In zebrafish, 17 out of 28 lines with heterozygous mutations in the rp genes are prone to develop malignant peripheral nerve sheath tumors (71,72). In Drosophila, haploinsufficiency in 64 of 88 cytoplasmic rp genes are proven to cause or likely cause Minute phenotype (73). It is still unclear why a small or a large subset of the rp genes are prone to the common haploinsufficiency phenotypes while others are not. Remarkably, the genes encoding L10a were excluded from such genes prone to the haploinsufficiency phenotypes, consistent with the idea that autoregulation of gene expression by AS-NMD can compensate for lack of one allele to some extent. A particular subset of the rp genes especially sensitive to changes in gene dosage may have acquired the autoregulation mechanisms in each organism.


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Direct in vivo Evidence for Increased Proliferation of CLL Cells in Lymph Nodes Compared to Bone Marrow and Peripheral Blood

Chronic Lymphocytic Leukemia (CLL) is a progressive malignancy of mature B-cells that involves the peripheral blood (PB), lymph nodes (LNs) and bone marrow (BM). While the majority of CLL cells are in a resting state, small populations of proliferating cells exist; however, the anatomical site of active cell proliferation remains to be definitively determined. Based on findings that CLL cells in LNs have increased expression of B-cell activation genes, we tested the hypothesis that the fraction of “newly born” cells would be highest in the LNs. Using a deuterium oxide (2H) in vivo labeling method in which patients consumed deuterated (heavy) water (2H2O), we determined CLL cell kinetics in concurrently obtained samples from LN, PB, and BM. The LN was identified as the anatomical site harboring the largest fraction of newly born cells, compared to PB and BM. In fact, the calculated birth rate in the LN reached as high a 3.3% of the clone per day. Subdivision of the bulk CLL population by flow cytometry identified the subpopulation with the CXCR4dimCD5bright phenotype as containing the highest proportion of newly born cells within each compartment, including the LN, identifying this subclonal population as an important target for novel treatment approaches.

Introduction
Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL) are Bcell malignancies that mainly affect the elderly. 1 CLL and SLL are considered different presentations of the same disease. 2, 3 CLL is defined as ≥5 000 monoclonal B-cells per μL in the peripheral blood (PB) with or without involvement of the lymphoid organs including the lymph nodes (LNs). In SLL, the affected cells are primarily in the LNs with ≤5 000 monoclonal B-cells per μL in the PB. Here we will refer to CLL as comprising both CLL and SLL.
Patients with CLL have a variable disease course with a third of patient's never needing treatment. In contrast, other patients need treatment soon after diagnosis and a subset of these only reach short remissions and undergo rapid decline and death thereafter. 4,5 Progressive CLL is often characterized by the usage of unmutated IGHV genes, high expression of CD49d, and genomic alterations that lead to a more rapid clonal expansion and inferior response to chemoimmunotherapy. 4,[6][7][8][9] CLL is characterized by a large population of resting cells which may be resistant to apoptosis and a smaller, but actively proliferating cell population. 10 The identification of the site of proliferation is of interest for understanding the process by which CLL progresses to more aggressive disease. Previous work using in vivo deuterium ( 2 H) incorporation estimated that between 0.1 and 1% of the CLL cells circulating in the PB are added to the population per day (referred to a "newly born" cells) and identified distinct CLL subpopulations that contain variable fractions of these newly born cells. [10][11][12][13] However, the anatomical compartment where active CLL cell proliferation occurs remains unknown. Proliferative or newly born CLL cells have been detected in PB, BM and LN, albeit of different clone sizes and with the use of different methodologies. [10][11][12][13] We recently showed that gene expression profiles of CLL cells in LNs are similar to those of activated, proliferating B-cells, while gene expression profiles of CLL cells present in the PB are similar to those of resting memory B-cells. 14, 15 We, therefore, hypothesized that the LN will be a critical site for CLL proliferation and progression.
Two cell surface membrane molecules have been particularly useful in identifying functionally different populations of CLL cells in the PB. These are the chemokine C-X-C motif receptor 4 (CXCR4), a chemokine receptor known to regulate cell trafficking, and CD5, a cell surface molecule expressed on normal T-cells, on a fraction of normal Blymphocytes, especially upon activation, and, characteristically, on CLL B-cells. Using the reciprocal densities of these two molecules on the surface of CLL cells obtained from the PB of patients who consumed 2 H 2 O, the CXCR4 dim CD5 bright fraction was identified as the population with the highest proportion of 2 H-labelled cells and has, therefore, been referred to as the proliferative subset. 16 Based on this data, we hypothesized that the CXCR4 dim CD5 bright population contains the cells that recently emigrated from the LNs into the circulating blood; however, the proliferative fraction of CLL cells in the LN remains to be characterized.
Here we sought to directly compare cellular growth rates of CLL cells collected simultaneously from patient matched PB, LNs, and BM using the 2 H in vivo labeling method and concurrent analysis of all three compartments. We now show conclusively that the proportion of newly born CLL cells is highest in the LN, compared to the PB and BM. Further, we directly demonstrate that the proliferative fraction of the clone is contained in the CXCR4 dim CD5 bright population in all three compartments.

Study Design
This study was designed as an auxiliary study to complement work on CLL within the Hematology Branch at the National Heart, Lung, and Blood Institute (Bethesda, MD, USA) and was registered as NCT01117142 with clinicaltrials.gov. The institutional review board approved these studies. Inclusion criteria were a diagnosis of CLL/SLL, age ≥18 years of age, absolute neutrophil count ≥1,000/μL, platelet count ≥50,000/μL. Patients using agents described to affect CLL cell biology or proliferation (including phosphodiesterase inhibitors, prednisone, cyclosporine, and green tea extract) were excluded. Lymph node biopsies were only obtained from patients with enlarged superficial lymph nodes. All study participants gave written informed consent. Diagnoses were made by established criteria. 17

H O administration and sample collection protocol
Patients were instructed to drink 42 ml (46 gm) of 100% 2 H 2 O (Sigma Aldrich, St. Louis, MO, USA) in the morning, noon, and evening for the first four days. Afterwards, patients drank 42 ml daily in the morning until they completed a total of four weeks. The goal was to achieve enrichment of 1-1.5% 2 H 2 O of total body water. Consumption of 2 H 2 O commenced on Day 1. A baseline PB sample was obtained prior to 2 H 2 O ingestion. Patients returned at scheduled intervals during and following the labeling period to donate blood for cell and serum analyses. BM and LN biopsies were performed on Day 13 ± 2. A labeling period of 2 weeks was chosen to provide sufficient time for equilibration of 2 H 2 O in total body water and incorporation of 2 H 2 O into DNA of proliferating cells. Saliva samples were collected to determine total body water enrichment. In addition, patients kept a daily log of 2 H 2 O intake which was discussed with each patient to assure compliance. No major unexpected side effects were encountered, although two patients reported a transient sense of lightheadedness and one patient reported a change in taste during the loading phase. These symptoms resolved spontaneously.

Cell preparation
PB mononuclear cells (PBMC) were isolated by density gradient centrifugation with Lymphocyte Separation Medium (MP Biomedicals, Santa Ana, CA, USA) and used fresh or after thawing frozen aliquots that had been cryopreserved in liquid nitrogen in 10% dimethyl sulfoxide and 90% fetal calf serum (Sigma Aldrich). Single cell suspensions from LN biopsies and BM aspirates were handled similarly. 14 CLL cells (>98% of CD19+ cells were CLL cells) were isolated using positive selection (CD19+ magnetic beads, Miltenyi, Auburn, CA, USA) and snap frozen.

Sorting into subpopulations with differential CXCR4 and CD5 expression and Ki67 staining
Isolation of cell fractions on the basis of expression of CXCR4 and CD5 was carried out as previously described. 16 Flow cytometry reagents were purchased from BD Bioscience, San Jose, CA, USA. Briefly, cells were incubated with murine anti-human mAbs: CD5-FITC (cat# 555352), CD19-APC (cat# 555415) and CXCR4-PE (cat #555974). After gating on CD19 + CD5 + events, cells were sorted with a BD FACSAria on the basis of intensity of CXCR4 and CD5 expression. Isolated cell pellets were stored at −80°C until analysis.

Measurement of body 2 H 2 O enrichment and 2 H incorporation into cellular DNA
2 H 2 O enrichment in plasma was determined at KineMed Inc. (Emeryville, CA, USA), using variations of two methods described previously. 19 Analysis of deuterium incorporation into DNA was determined using methods previously described. 20,21 Briefly, cells were disrupted by incubation with proteinase K and filtered to remove cell debris. DNA retained on the filter was hydrolyzed. Clean up, derivatization, and isotope ratio mass spectrometry (IRMS) was carried out as previously detailed. 21 Sorted subpopulations with differential CXCR4 and CD5 membrane densities were analyzed by gas-chromatography/mass spectrometry (GC/MS) as previously described. 16

Body 2 H 2 O enrichments to estimate precursor enrichment
Body water enrichment data were evaluated using a single compartment model in which the fit parameters were the subject's total body water (TBW) volume and body water turnover. In this model, TBW is considered a single, homogeneous, well-mixed pool at steady-state, into which heavy water is introduced in known amounts and at known intervals. The model parameters, TBW and body water turnover (kw), were determined by least squares fit to the measured body water enrichment data obtained throughout the study. These data were analyzed with SAAM II software (Simulation, Analysis, and Modeling; SAAM Institute, University of Washington, Seattle, WA, USA). In this way, a continuous time course of body water enrichment was obtained for each subject. The average heavy water exposure during the labeling period relates to the amount of isotope that could be incorporated into newly synthesized DNA. To calculate average body water exposure for each sample timepoint, the body water enrichment time course modeled as above was integrated up to that timepoint (using the trapezoid rule) and represents the time averaged precursor enrichment (p) at each timepoint.

H enrichments in DNA and calculation of fractional DNA synthesis
In order to calculate the newly born CLL cell fraction for each timepoint from GC/P/IRMS data, atom percent excess (APE) was first calculated from measured 2 H-1 H: 1 H-1 H abundance ratios in the GC peaks corresponding to the dR derivative. The maximal or asymptotic amount of 2 H incorporation (APE*) for fully turned-over (100% new) DNA was calculated as: APE* = p × 5.5/59, where p is the average precursor enrichment (as above) and the numerical conversion ratio corresponds to the number of hydrogen atoms in dR accessible to 2 H 2 O labeling, divided by the number of hydrogen atoms in the derivative. Fractional DNA synthesis (F) was then calculated using the ratio of the measured APE to the APE*.
For the DNA samples analyzed for isotopic enrichment by GC/MS, F was calculated from the deuterium enrichment in dR (EM1) and the time-averaged p using a mathematical relationship previously described (F = EM1/EM1*, where EM1* = 3.025 × p+0.0014). 21 A daily rate of appearance of newly born CLL cells (k) was obtained by fitting F to the exponential rise-to-plateau equation [F = 1-exp (-k t)].

Statistics
To compare measurements in patient samples from PB, LN, and BM either across time or compartment, a paired Student's t-test was used unless otherwise stated (Prism5, GraphPad, La Jolla, CA, USA). Values of P<0.05 were considered statistically significant.

H incorporation into the DNA of CLL cells is higher in LN compared to PB and BM
Fifteen patients ingested 2 H 2 O for 2-4 weeks; 12 had CLL and three SLL. Collectively, we will refer to the tumor cells as CLL cells. The demographic and baseline disease characteristics of the patients are summarized in Table 1. One patient was previously treated; the other 14 patients were treatment naïve, eight had stable disease, and seven had active or progressive disease at the time of 2 H 2 O labeling. Seven patients had active disease and initiated treatment within a median of 1.6 months from starting 2 H 2 O intake. The content of 2 H 2 O in total body water was measured weekly. Enrichment of 2 H 2 O in the desired range of 1 to 1.5% of total body water was reached by day 7 and maintained throughout the labeling period ( Figure 1a). Excisional LN biopsies and/or BM aspirates were obtained on day 13. PB was sampled at baseline and on day 13 and when possible on day 28 and day 35. CLL cells were purified using positive selection, and 2 H incorporation into the cellular DNA was measured by mass spectrometry. Based on the level of 2 H incorporation into the DNA of CLL cells, the newly born CLL cell fraction, i.e. cells generated during the labeling period, was determined for each sample and timepoint. The newly born CLL cell fraction differed significantly between the three anatomic sites (P<0.01, by repeated measures ANOVA). At day 13, the fraction of newly born CLL cells was largest in the LN at 33% followed by 23% in PB and 10% in BM (Figure 1b). Thus, in some patients up to one third of the tumor burden in the LN at day 13 consisted of cells born within the preceding two weeks.
In a previous study the "birth rate" of cells was estimated from the fraction of newly born CLL cells present in PB samples. 10 Here we concurrently measured the fraction of newly born CLL cells in three different anatomic compartments. Because the fraction of newly born CLL cells at any one site is determined by the birth rate at the given site and the rate and direction of exchange of newly born cells between compartments we refer to k as the % newly born cells added to the total population in any given compartment per day. The median value of k in the PB was 0.56 (range 0.08 -2.11), in the LN 1.27 (0.42 -3.32), and in the BM 0.51 (range 0.18 -0.96). Across different patients and compartments k was highly variable (Table 2). In twelve patients PB and LN samples were obtained concurrently and k was significantly higher in LN than PB in all except on patient (P=0.03; Figure 2a). In eight patients PB and BM samples were obtained concurrently; k was not significantly different across all eight samples (P=0.2; Figure 2b) and in four of eight patients, k was actually lower in the BM than in PB. In five of six patients with concurrent BM and LN samples k was higher in LN than BM but the overall difference did not reach significance (P=0.07; Figure 2c). In patients who completed four weeks of 2 H 2 O ingestion, we obtained additional PB samples on day 28 and day 35 (Table 2). Consistent with continuous cell proliferation and 2 H 2 O incorporation into cellular DNA, the fraction of newly born cells increased from day 13 to day 28 (data not shown). However, the rate at which newly formed cells appeared in the PB was constant over time, with k = 0.51, 0.48, and 0.52 %/day on days 13, 28, and 35, respectively. Further, in individual patients, there was very little variability of k on day 13 compared to day 28 (Figure 2f). Thus, the birth rate of CLL cells, while highly variable between different patients, appears to be quite steady in the individual patient, at least over the timeframe studied.

The fraction of newly born cells (k) is highest in the CXCR4 dim /CD5 bright subset
Previous cell kinetic studies using 2 H 2 O labeling showed that a CXCR4 dim /CD5 bright subpopulation in the PB contained a larger fraction of newly formed cells than the bulk of the CLL cells. 16,22 These CXCR4 dim /CD5 bright cells are thought to be recent emigrants from tissue sites, in particular from LNs. Here, we extended this analysis to LN and BM samples. CLL cells from PB, LN, and BM were sorted into three subpopulations as previously described: 16,22 the CXCR4 dim /CD5 bright (db) "proliferative", the CXCR4 int / CD5 int (ii) "bulk", and the CXCR4 bright /CD5 dim (bd) resting/re-entry subsets (Figure 3a). The CXCR4 dim /CD5 bright subset had the highest mean k in all three compartments and contained at least 3-times as many newly born cells than any of the other fractions within the same anatomic site (P<0.0001 in PB and LN; Table 3, Figure 3b). Interestingly, k in the CXCR4 dim /CD5 bright subset from LN and PB was quite comparable at 3.6 and 3.2%/day, respectively. At this rate, the whole proliferative fraction (CXCR4 dim /CD5 bright ) is being replaced with newly formed cells every month. In contrast, only between 0.5 to 1% newly formed cells are added each day to the CXCR4 int /CD5 int and the CXCR4 bright /CD5 dim subsets that therefore contain mostly older cells (estimated greater than 70% of cells older than 1 month). Compared to LN and PB, k for each subset was the lowest in the BM.

Birth rate of CLL cells and disease characteristics
Next, we correlated the growth rate of the LN populations with clinical characteristics and prognostic markers. Of the 13 patients who underwent LN sampling, seven had active disease and six had stable disease according to IWCLL criteria (Table 1, Figure 4). 17 Patients with active disease had the highest growth rate, on average 1.6% of the LN clone per day, which was more than twice the rate in patients with stable disease (Figure 5a, P=0.001), while the growth rate of the PB and BM was not statistically different between patients with active and stable disease. There was an inverse correlation of the growth rate in LN populations with lymphocyte doubling time (ρ=−0.83; P=0.008; Figure 5b) and the time to first treatment (r=−0.685; P=0.01, Figure 5c), consistent with increased proliferation in the LN as a driver of active disease and the requirement for early treatment. There was also a trend towards higher growth rate in patients with IGHV unmutated CLL (Ig-unmutated) compared to patients with IGHV mutated (Ig-mutated) disease (P=0.07; Figure 5d). CCL3 and CCL4 are chemokines secreted by activated CLL cells in the LN and high CCL3 serum concentrations have been associated with more rapid disease progression. 14, 23 We therefore measured CCL3 and CCL4 serum levels in our patients. While there was a trend for higher CCL3 serum levels in patients with Ig-unmutated CLL there was no significant correlation between k in LN and CCL3 (r= 0.36; P= 0.23) or CCL4 (r= 0.295; P= 0.32) serum levels (Supplemental Data).
Four types of cytogenetic abnormalities routinely assessed by FISH are associated with clinically meaningful differences in survival. In order of associated shortest to longest median survival time, these are 17p deletion, 11q deletion, trisomy 12, and 13q deletion. 24 The presence of 17p deletion was associated with a higher growth rate of the LN population compared to 11q deletion, trisomy 12, and 13q deletion (P=0.01; Figure 5e). The three patients with 17p deletion had higher than median growth rates and active disease, while patients with trisomy 12 or 13q deletion had variable growth rates and some of these patients had stable disease. There was no correlation between growth rates and CD38 expression, age, or gender. The growth rate in the LN for patients with SLL was virtually the same as in patients with CLL.

Discussion
In this report, we identify that the major anatomic site of the proliferating cells in a CLL clone is the LN, and that the primary proliferative fraction of these cells expresses a CXCR4 dim /CD5 bright phenotype. This is consistent with gene expression profiling studies showing that the LN compared to PB and BM is the site for upregulation of gene signatures associated with B-cell receptor activation and proliferation of CLL cells. 14 The higher growth rate of CLL cells in the LN compared to PB and BM measured by in vivo labeling of newborn cells with 2 H is further supported by the finding that expression of the cell cycle marker Ki67 is highest in CLL cells in the LN. In fact, the average Ki67 positive fraction in LN was five times the fraction in the PB supporting the view that active clonal proliferation occurs primarily in the LN and this is the site from which newborn cells enter the PB. While we cannot estimate the recirculation capacities of CLL cells, the migration from the LN to PB appears to follow first order kinetics and is dependent on the rate of appearance of new cells (k) in the LN. This is the first report using 2 H in vivo labeling to identify the growth rate of CLL populations concurrently in all three major anatomic compartments. The highest rates of cell growth and proliferation were found in the LN compared to BM and PB. The presence of CLL cells with increased birth rates in the PB and BM is consistent with the finding of similar, but less pronounced expression of gene signatures associated with CLL cell activation and proliferation in the BM compared to the LN and suggests that although the BM is predominately inhabited by CLL cells in a resting state, a small fraction of recently born cells is also found here that may be made up of recent emigrants from LN or of cells having divided in situ. Due to the technical limitations of BM aspiration, representation of all BM resident cells in the aspirate cannot be ensured, and we cannot rule out that BM cells bound more tightly to the microenvironment and, therefore, not represented in the aspirate have a higher birth rate. However, our finding of low CLL cell turnover in the BM is in line with previous work by van Gent and colleagues. 13 Of note, k in the PB of HW07, the only patient showing a higher k in PB than LN, was nearly identical on day 13 and day 28. While we cannot provide a definitive explanation for the larger fraction of newly born cells in the PB compared to LN, it is noteworthy that this was a patient with predominantly nodal presentation and an ALC of only 2,000 cells per μl in the blood. It is, possible that the enrichment of newly born cells in the PB may be due to different trafficking and/or homing properties of subpopulations in this patient. With disease progression and/or after having received multiple treatments, the cellular kinetics that we observed may be altered. This work, therefore, does not exclude the possibility that CLL cells may proliferate at a higher rate in the PB or BM in more aggressive disease or relapsed/refractory disease.
Other studies determined cell proliferation in CLL cells using 2 H in vivo labeling. The majority of these studies analyzed bulk populations in the PB and estimated the CLL cell growth rate at 0.1 to 1% per day. 10,11,13 Most recently, a proliferative fraction of CLL cells primarily among CXCR4 dim /CD5 bright cells was identified in PB. 16 Our results confirm the presence of this proliferative population in the PB and extend the first observation by identifying the presence of phenotypically similar populations in the LN and BM that also have a higher growth rate than the other subpopulations in the same compartment (Figure 3). CXCR4 is dynamically regulated in response to antigen stimulation and CXCL12 ligation and thus identifies cells that have recently been exposed to CXCL12 within the tissue compartment or have recently been stimulated. 14,16,[25][26][27] Notably, the % newly born cells appearing in the CXCR4 dim CD5 bright populations in PB and LN was not statistically significantly different, albeit still somewhat higher in the LN than in the PB. The similar size of the fraction of newly born cells among the proliferative fraction in PB and LN is not unexpected, based on current thinking. In the prevailing model of CLL cell proliferation, 16 the CXCR4 bright CD5 dim fraction of cells in a resting state. When these cells are activated, CD5 is upregulated and CXCR4 is internalized. These cells, now bearing a CXCR4 dim / CD5 bright phenotype are released from the LN and migrate into the PB. Eventually these cells become more quiescent leading to downregulation of CD5 and renewed surface expression of CXCR4 that could increase the likelihood of a return to the LN. In line with this view, it has previously been shown that CLL cells expressing activation-induced deaminase (AID) are almost exclusively found in the CXCR4 dim CD5 bright population; further suggesting these cells represent the proliferative subclone. 28 Our observation that the CXCR4 dim /CD5 bright population contains the highest proportion of newly born cells in any compartment suggests that the interchange between the immunophenotypically defined subpopulations, for example from proliferative to resting/reentry populations, is quite slow. On the other hand, the minimal difference in the proportion of newly born cells between the CXCR4 int CD5 int and the CXCR4 bright CD5 dim subpopulations, would be consistent with rapid equilibration of cells between these two populations. Alternatively, the CXCR4 dim CD5 bright phenotype could identify a subclonal population that is distinct in its life-cycle from other subpopulations and that could disproportionately contribute to clonal expansion and more fluidly traffic between compartments. Studies are ongoing to further dissect the transcriptomic and functional characteristics of the different subpopulations.
The more progressive disease course associated with the absence of somatic mutations in the IGHV gene, referred to as Ig-unmutated CLL, compared to Ig-mutated CLL is well established. 1,4 The exploratory analyses of CLL growth rates in patients stratified by IGHV gene mutations status measured higher CLL growth rates in the LNs of patients with Igunmutated CLL than in patients with Ig-mutated CLL (Figure 4c). Further, high growth rates in the LN were associated with active disease, faster lymphocyte doubling time, and shorter time to first treatment than in CLL with low growth rates. These data are consistent with the prior observation of increased BCR signaling in the LN of Ig-unmutated compared to Igmutated CLL and the shorter time to treatment in patients with higher tumor proliferation in the LN and the presence of more aggressive disease in the PB. 10,14 The 15 patients studied here overall reflect the heterogeneity of the CLL patient population quite well, including low to high risk Rai stage and representing both IGHV subtypes equally. At study entry, the time from diagnosis ranged from 1 to 11 years and 7 (47%) patients had active disease requiring treatment. While limited by a small sample size, our findings are statistically sound and consistent with previous gene profiling studies that utilized greater numbers of patients.
Our results identify the LN as the primary anatomical site of CLL cell proliferation from which newly born cells are released into other compartments. Our data advance the understanding of CLL cell trafficking between the LN and the periphery. Further, the subpopulation of CXCR4 dim /CD5 bright cells contains the proliferative fraction of CLL cells in all three anatomic compartments, identifying this population as a valuable target for novel treatment strategies.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.    Table 3. Paired T test was used to compare k between cellular fractions within each anatomic compartment.      Table 2 Rate of appearance of new cells k (%/day)  Table 3 Rate of appearance of new cells k (%/day) for sorted fractions