extfieldsofstudy: Medicine Biology

text:
Transcriptional profiling of circadian patterns of mRNA expression in the chick retina.

Previous transcriptome analyses have identified candidate molecular components of the avian pineal clock, and herein we employ high density cDNA microarrays of pineal gland transcripts to determine oscillating transcripts in the chick retina under daily and constant darkness conditions. Subsequent comparative transcriptome analysis of the pineal and retinal oscillators distinguished several transcriptional similarities between the two as well as significant differences. Rhythmic retinal transcripts were classified according to functional categories including phototransductive elements, transcription/translation factors, carrier proteins, cell signaling molecules, and stress response genes. Candidate retinal clock transcripts were also organized relative to time of day mRNA abundance, revealing groups accumulating peak mRNA levels across the circadian day but primarily reaching peak values at subjective dawn or subjective dusk. Comparison of the chick retina transcriptome to the pineal transcriptome under constant conditions yields an interesting group of conserved genes. This group includes putative clock elements cry1 and per3 in addition to several previously unidentified and uninvestigated genes exhibiting profiles of mRNA abundance that varied markedly under daily and constant conditions. In contrast, many transcripts were differentially regulated, including those believed to be involved in both melatonin biosynthesis and circadian clock mechanisms. Our results indicate an intimate transcriptional relationship between the avian pineal and retina in addition to providing previously uncharacterized molecular elements that we hypothesize to be involved in circadian rhythm generation.

Circadian biological rhythms are a pervasive property of most multicellular organisms, most eukaryotic microorganisms, and at least some prokaryotic taxa. It has been known for some time that circadian rhythms are genetically determined (1). Circadian traits such as free-running periods () and phase-angle (⌿) to entraining light cycles can be selected for, and mutations for these traits have been identified, leading to the isolation and characterization of molecular clock compo-nents (2,3). In the last 10 -15 years, the mRNA and protein products of many of these "clock genes" have been shown to oscillate with a genotype-specific period, and their protein products have been shown to interact in vitro (4 -7).
These rhythmic profiles have suggested a generalized model for circadian rhythm generation that involves the transcription, translation, and feedback of these clock gene products on their own transcription. The proposed interlocking transcriptional/translational feedback loops are comprised of "negative elements" and "positive elements" which interact to drive overt rhythmicity (4 -7). Briefly, the negative elements in mammals are encoded by the period genes (per1-3), cryptochromes (cry1 and -2), and Rev-Erb␣. These are transcribed in response to the dimerization of the positive elements, which include clock (clock) and the Bmals (Bmal1, 2) and their subsequent binding to E-boxes in the promoter regions of several genes. The negative element transcripts are then translated, oligomerize, and re-enter the nucleus where they inhibit their own and other gene transcription, closing the feedback loop.
In vertebrates, circadian organization also relies on discrete neuroendocrine structures that drive and/or entrain downstream rhythms of biochemistry, physiology, and behavior. These structures include the hypothalamic suprachiasmatic nucleus (SCN), 1 the pineal gland, and the retina. In mammals, the SCN serves as a master pacemaker, responsible for coordinating all downstream oscillators and rhythmic processes. In birds and many other non-mammalian vertebrates, however, the pineal gland, retina, and the avian homologue of the SCN are equally important in regulating overt rhythmicity. The avian pineal gland and retina share several key processes; 1) both are directly photoreceptive, 2) both contain circadian oscillators, and 3) both rhythmically synthesize and release the indoleamine neurohormone melatonin.
At the systems level, the retina functions integrally with the pineal gland and the SCN such that these oscillatory structures feedback and influence each other (20 -23). Under several experimental circumstances loss of the retinal clock in turn disrupts and/or abolishes the clock of the organism or output processes (24 -27). The retina also is responsible for integrating and relaying photic information to the SCN via retinal ganglion cells and the retinohypothalamic tract. This tract serves as the major pathway for entrainment of the SCN in mammals (28,29) and has been an area of great focus in the search for the photopigment(s) existing in retinal ganglion cells that serves as the entraining photoreceptor of the system.
Our previous transcriptome analyses (30) identified candidate molecular components of the avian pineal clock. Here, we determine candidate retinal clock transcripts under light dark and constant darkness conditions to better understand the molecular organization of the avian system. Subsequently, a comparative transcriptome analysis of the two data sets determined candidate molecular machinery regulating the circadian system of chicks within both pacemakers and circadian oscillators.

EXPERIMENTAL PROCEDURES
Experimental Animals-Chicks were obtained from Hyline International (Bryan, TX) and housed under a lighting schedule of 12 h of light and 12 h of darkness (LD 12:12) for 7 days with food and water continuously available. For microarray hybridizations chicks (n ϭ 3/time point/experiment) were sacrificed by decapitation, and their retinas were dissected and pooled. Two separate retinal RNA sampling cycles were performed for array hybridizations. All DD dissections were performed using an infrared viewer. Animals were treated in accordance with Public Health Services guidelines; these procedures have been approved by the Texas A&M University Laboratory Animal Care Committee (AUP #2001-163).
Microarray Production and Hybridization-The cDNA microarrays were prepared as previously described (30) based upon ϳ4500 PCR products from each of the two pineal cDNA libraries constructed, for a total of ϳ9000 ESTs. Briefly, the cDNA in plasmids from each library were PCR-amplified using flanking primers (SK and T7), purified by ethanol precipitation using ammonium acetate, and placed in the wells of 96-well plates at a concentration of 50 g/ml in 3ϫ SSC (1ϫ SSC ϭ 0.15 M NaCl and 0.015 M sodium citrate). A GeneMachines OmniGrid microarrayer equipped with 8 Telechem SMP3 pins was used to spot the samples onto poly-L-lysine-coated slides (CEL Associates) in duplicate. Slides were stored desiccated at room temperature until their subsequent use. Before hybridization, the dried spots were hydrated gently over a steaming water bath and snap-dried, and the DNA was UVcross-linked using a Stratalinker (Stratagene).
Total RNA was harvested using Trizol Reagent (Invitrogen) every 4 h for 1 day in a light-dark cycle of 12 h light and 12 h darkness (12:12; lights on 0600 Central Standard Time; lights off 1800 Central Standard Time) and for 1 day in continuous darkness (DD). Times of sampling began 2 h after lights-n (zeitgeber time, ZT2) and continued every 4 h hence (ZT6, ZT10, ZT14, ZT18, ZT22). When lights would have normally turned on, the timer was disabled, and birds were placed in DD. They were then collected again every 4 h and designated circadian time (CT) CT2, CT6, CT10, CT14, CT18, and CT22. This sampling procedure was performed on two separate occasions. A total of four experimental microarray hybridizations were conducted for each time point, two from each respective biological sampling procedure.
The total RNA samples were then amplified to produce aRNA using a MessageAmp Kit (Ambion). Randomly primed fluorescent probes were produced from aRNA (amplified RNA) samples using a Genisphere 3DNA Array 900MPX expression array detection kit. The fluorescent dye on probes derived from the experimental aRNA was Cy5, whereas the dye on control probes was Cy3. In all experiments the control sample was derived from retina harvested at midnight (ZT18) under LD conditions.
Hybridizations and washes were conducted as suggested by Genisphere. The labeled arrays were scanned in an Affymetrix 428 array scanner. The images obtained were subsequently analyzed by GenePix-Pro software (Axon Instruments).
Data Analysis-Data files were then further analyzed by GeneSpring 6.1 (Silicon Genetics). GeneSpring allows the data to be normalized in a variety of ways, allows the assignment of parameters and interpretations, and then allows the data to be filtered to determine differential expression. The current analysis used intensity-dependent LOWESS normalization. Intensity-dependent normalization is just one technique used to eliminate dye-related artifacts in two-color experiments such as this. At each time point the results for each gene were reported as an average obtained from four slides. The data are reported as the normalized ratio of Cy5 (experimental) to Cy3 (control at ZT18). Thus, the data reported for each time point are relative to the abundance of the same gene at midnight (ZT18).
The criteria used to define rhythmic genes were those transcripts with a 2.0-fold or higher amplitude change in mRNA levels over a 24-h period relative to the gene respective mRNA abundance level at ZT18 in addition to exhibiting this oscillation in 3 of 4 hybridizations performed. Analysis at the 1.5-fold level was also performed, but not to the extent of examination as the 2.0-fold data set. Those gene sets that were determined to achieve the 1.5-and 2.0-fold criterions were analyzed statistically by one way analysis of variance, with p Ͻ 0.05 considered significant. Only statistically significant changes will be reported here. However, the entire dataset is available from the authors. 2

RESULTS
Transcriptome Analysis-Of the ϳ9000 cDNAs represented on the microarray, 546 classified transcripts oscillated with 1.5-fold amplitude, and 175 classified transcripts oscillated with 2-fold or greater amplitude in a light-dark cycle (Supplemental Tables 1 and 2). Upon examination of these transcripts under conditions of constant darkness, the number of oscillating transcripts observed was reduced to 383 at 1.5-fold and to only 49 at 2.0-fold or better (Supplemental Table 3 and 4). This percentage of rhythmic transcripts is similar to those found in the chick pineal gland (30) and other experimental analyses of rhythmic processes using cDNA and/or oligonucleotide arrays (31)(32)(33)(34). We restricted our most detailed analyses to those transcripts that varied by 2.0-fold or more.
Functional Categories of Rhythmically Expressed Transcripts-Oscillating retina transcripts in LD and DD were classified according to proposed function. Components of the melatonin generating enzymatic cascade were represented, as were many chick orthologs associated with mammalian circadian clock function. Furthermore, functional analysis revealed transcripts in several broad categories that were also rhythmically expressed. Functional categories were divided into 20 categories: intermediary metabolism (A), development (B), melatonin biosynthesis (C), neuronal associated (D), disease related (E), hormones/growth factors (F), cell cycle/cell death (G), carrier proteins/transport/circulation (H), cytoskeleton/microtubule-associated (I), cell signaling (J), cell adhesion (K), RNA synthesis/stability (L), circadian clock (M), photoreception/phototransductive/visual cycle elements (N), ribosomal proteins/ translation (O), stress response/host defense/chaperone (P), transcription factors (Q), protein modification (R), receptors (S), DNA synthesis/replication/binding (T), and miscellaneous functions that don't readily fit into a single category above (U). The stress response/chaperones and cytoskeletal classes under LD conditions had the highest number of oscillating transcripts, with 28 and 23, respectively. In DD the stress response/ chaperones again had the largest classification, with eight, followed by the cytoskeletal, carriers, phototransduction, and ribosomal clusters, with five members in each. These functional classifications are indicated in Fig. 1, A and B, and also Supplemental Tables 1-4.
Circadian Phase Analysis-Initial analysis of daily and circadian phase relationships revealed rhythmic transcripts that exhibited a diverse set of phases in the LD cycle. However, retinal transcripts rhythmically expressed in DD either accumulated primarily during the day or peaked during the night. These were clustered using a K-means clustering algorithm (GeneSpring) according to the phase of mRNA abundance. The clustering analysis revealed three primary clusters of peaking transcript abundance at CT22-2, CT6, and CT14 (Fig. 2). For each cluster a representative trace is shown indicating the average profile of all oscillating transcripts in each respective cluster. Cluster 1, which composed 20% of the unique DD transcript set, is indicative of transcripts peaking at approximately the onset of subjective dawn CT-22-CT-2; Cluster 2, 49% composed of the unique DD transcripts, at mid-subjective day CT-6; Cluster 3, composed 31% of the unique DD transcripts, at early subjective night CT-14. This distribution of phases is similar to previous findings of other groups (31)(32)(33)(34).
Melatonin Biosynthesis-As has been previously shown, the mRNAs encoding enzymes involved in melatonin biosynthesis were rhythmically expressed on a daily and circadian basis. Under LD conditions the data indicated a rhythm in tryptophan hydroxylase mRNA such that levels were highest in the early night (ZT14), and hydroxyindole-O-methyltransferase was highest at midnight (ZT18). AANAT mRNA exhibited a low amplitude rhythm that was highest during the night as well (Fig. 3A). These patterns corresponded favorably with Northern blot data employing radioactive probes derived from the microarray cDNAs corresponding to each respective gene (Fig. 3B). The rhythm in tryptophan hydroxylase persisted in DD with similar phase angles, whereas hydroxyindole-O-methyltransferase and AANAT mRNA were not significantly rhythmic in DD at the 2.0-fold criterion (Fig. 3A).
Clock Genes-Several mRNAs encoding putative orthologs of genes associated with circadian clock function were identified in the dataset. These include putative negative elements cryptochrome 1 (cry1), cryptochrome 2 (cry2), period 2 (per2), and period 3 (per3) (Fig. 4A). Under both LD and DD conditions cry1 and per3 exhibited rhythmic mRNA profiles, such that peak cry1 mRNA levels occurred during the midday (ZT6) in LD and mid-subjective day (CT6) in DD, and peak per3 mRNA levels occurred during the late (ZT22) night and subjective night (CT22). Although neither cry2 nor per2 mRNA levels were rhythmic in LD, per2 mRNA levels expressed a modest increase at CT10 in DD. These profiles and their phases were confirmed by Northern blot analyses (Fig. 4B). The putative positive elements clock, bmal1, and bmal2, as well as casein kinase 1 epsilon (ck1E) were examined. Analysis of the putative positive elements yielded a low amplitude rhythm in LD for bmal1 and bmal2, peaking in the early night, ZT14, whereas clock was not rhythmically expressed. In addition, no rhythm in ck1E mRNA abundance was observed. In DD, clock, bmal1, and bmal2 were rhythmically expressed, reaching peak mRNA abundance at CT10 -14 (Fig. 4C).
Retinal and Phototransduction Transcripts-Several components of the photoreceptive/phototransduction/visual cycle pathway were examined on the microarray, yielding many rhythmic profiles under LD and DD (Fig. 5). Because the microarray was produced from cDNA libraries derived from chick pineal glands, it is important to point out here that retinaspecific sequences (genes expressed in the retina but not the pineal gland), such as rhodopsin, are not represented here. Nonetheless, several genes associated with visual transduction were analyzed including melanopsin (Opn4) (35) and rod ␥-subunit phosphodiesterase 6 (Pde6) (36) as well as opsins and binding proteins associated with visual cycle function, such as peropsin (Rrh) (37), retinal G-protein-coupled receptor opsin (Rgr) (38), and retinal pigmented epithelium 65 (Rpe65) (39). The mRNA for all of these genes was expressed rhythmically under both LD and DD conditions with the exception of melanopsin. Although melanopsin was expressed predominantly in the late day through night in LD, there was no apparent circadian pattern of expression in DD. Additional potential elements of phototransduction and visual cycle function that were examined but found to be not significantly rhythmic under our criteria include transducin ␥-subunit (40) Tables 2 and 4. retinaldehyde-binding protein (CRALBP) (42), and retinal short chain dehydrogenase/reductase 2 (43).
Retinal Transport-The vertebrate visual cycle process involves at least three different retinal cell-types; that is, the photoreceptors, pigmented epithelial cells, and Muller glia. Thus, the problem of transported retinaldehyde and by-products from one compartment to another and from the periphery into the retina is likely solved by transport proteins. One of these is purpurin (44), whose mRNA is rhythmically expressed in the retina. Purpurin was expressed highly during the early day and late night in LD and early subjective day and late subjective night in DD (Fig. 6A). Another retinal transport molecule is transthyretin, which was found to be rhythmically expressed in the pineal gland (30), was not consistently rhythmic in the retina under constant conditions.
Translational Control and Protein Trafficking/Modification-A number of ribosomal and ribosome-associated proteins were rhythmically expressed in both LD and DD. The 40 S ribosomal proteins S3, S5, S18, and S27 as well as 60 S ribosomal proteins L10 and L18 were expressed predominantly during the day with a 2.0-fold rhythm. Of these, S27, L10, and L18 were expressed rhythmically with a 2.0-fold or higher amplitude such that expression was highest during subjective day in DD (Fig. 6B). In addition, chick elongation factor 2 (EF-2) and ribosomal protein L11 were expressed rhythmically in both LD and DD such that mRNA was highest in late day in LD and late subjective night in DD.
A large number of mRNA species associated with proteolytic proteasomal activity were rhythmically expressed in LD. These included several ubiquitin sequences as well as sequences associated with ubiquitin modification and several protein phosphatases and carboxypeptidase M (Supplemental Table 1). Of these, only carboxypeptidase M persisted in its rhythmicity in DD at the 2.0-fold criterion, peaking at CT14 in the early subjective night. However, most of the ubiquitin-associated sequences persisted in DD at the 1.5-fold criterion, peaking during the subjective day. Intermediary Metabolism-Many mRNA species associated with glycolytic, lactate fermentation, and electron transport processes were expressed rhythmically in LD at the 2.0-fold criterion (Supplemental Table 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglucose isomerase, and lactate dehydrogenase were all expressed predominantly in the mid-to late day (ZT6 -10). Additionally, cytochrome b and NADH dehydrogenase subunit 4 were expressed highly at midday (ZT-6). ATP synthase ␤-subunit, ATP synthase Hϩ transporter, and NADH dehydrogenase (ubiquinone) 1␣ were expressed predominantly at early day (ZT2), and cytochrome c oxidase subunit II was expressed at early night (ZT14). In DD several of these oscillations failed to persist, although GAPDH and cytochrome c oxidase subunit II were expressed rhythmically at the 2.0-fold criterion, with the former expressed in the late subjective day and the latter expressed in the early to late subjective night (Fig. 6C). However, most of the electron transport system genes were expressed rhythmically in DD at the 1.5-fold criterion (Supplemental Table 3).
Stress Response/Host Defense/Chaperone-Many mRNA species associated with stress response were found to be rhythmic in LD cycles (Supplemental Tables 1 and 2). These included cathepsin and cystatin, cytochrome p450, the aryl hydrocarbon receptor, several heat shock proteins, and several genes associated with histocompatibility. Of these, cytochrome P450, 3 heat shock proteins (HSP8, HSP90, and a 70-kDa cognate heat shock protein ATPase), 3 antigen identification genes (␤2-microglobulin, BM88 antigen, major histocompatibility complex class II associated invariant chain proteins), and the aryl hydrocarbon receptor persisted in DD at the 2.0-fold criterion (Supplemental Table 4). DISCUSSION Both the retina and pineal gland have been identified as circadian pacemakers in the avian circadian system. Of course, both are photoreceptive structures, containing both common and tissue-specific photopigment and phototransductive cascades. For example, both tissues express melanopsin, peropsin, retinal G-protein-coupled receptor opsin, cryptochromes, transducins, and cGMP phosphodiesterase among other phototransductive sequences. Furthermore, photoreceptor cells in both tissues rhythmically synthesize and, at least in some species, release the indoleamine hormone melatonin, such that melatonin is synthesized during the night in LD cycles and during the subjective night in DD in both tissues. This circadian secretion of melatonin accounts for most if not all of the pineal and retina system level functions. However, the relative roles of the pineal and retina clock vary among avian species. For example, in oscine passerine birds such as the house sparrow, Passer domesticus, pinealectomy abolishes circadian patterns of locomotor activity and of physiological functions, such as body temperature (45,46), whereas complete enucleation has little effect on either entrainment or circadian patterns of activity (47). Conversely, pinealectomy of juvenile and adult domestic fowl has little effect on overt behavioral rhythms of activity and feeding (10), whereas enucleation abolishes locomotor rhythms A, retinal transport. Purpurin mRNA is rhythmically expressed in the retina with peak accumulation during the early day and late subjective night in DD. B, ribosomal proteins. S27, L10, and L18 in addition to chick elongation factor 2 (EF-2) and L11 were expressed rhythmically. C, intermediary metabolism. GAPDH and cytochrome c oxidase (COX) subunit II were also expressed rhythmically. (48). Even so, although pinealectomy has no effect on overt behavioral rhythms, the surgery abolishes circadian patterns of electroretinogram a-and b-wave function (10), and administration of exogenous melatonin affects both electroretinogram and visually evoked potentials in chicks and pigeons, Columba livia (10,49,50). In some species the retina and pineal contribute to overt rhythms equally. Neither pinealectomy nor enucleation alone abolishes locomotor rhythms in Japanese quail, Coturnix japonica, and pigeons, but the two surgeries combined abolish behavioral rhythms in both species (20,51). Thus, both the pineal gland and retina are photoreceptive, circadian oscillators that affect behavioral and physiological rhythms via the rhythmic production of melatonin. In chicks, therefore, they represent two sets of circadian pacemakers that reside in the same organism, which one would expect to employ identical molecular mechanisms to generate and regulate their rhythmic functions.
In the chick pineal gland tryptophan hydroxylase and AANAT mRNA are expressed rhythmically, such that levels are high during the night and low during the day in LD, whereas hydroxyindole-O-methyltransferase mRNA is expressed higher during the day than during the night. All three of these rhythms persist in DD (30,56).
In contrast, in the retina, tryptophan hydroxylase, AA-NAT, and hydroxyindole-O-methyltransferase mRNA are regulated on a daily basis such that tryptophan hydroxylase and hydroxyindole-O-methyltransferase are high at early to late night and AANAT is high at midnight (56,57). In DD, hydroxyindole-Omethyltransferase becomes arrhythmic, whereas tryptophan hydroxylase and AANAT mRNA continue to accumulate with similar phases as in the LD cycle, albeit with a lower amplitude (19,56). These patterns of expression are confirmed in the present study in both the microarray data and in Northern blot analyses of the same mRNA extracts (Fig. 3, A and B), demonstrating the validity of this microarray approach and punctuating the observation that, at the mRNA level, melatonin biosynthesis is regulated differentially in the chick pineal and retina.
The mechanism by which rhythmic melatonin biosynthesis is regulated has been assumed to include the transcription, trans-lation, and post-translational activation of chick orthologs of mammalian clock genes. Many of these orthologs have been identified and characterized, at least within the pineal gland (14,15,30,60,61). These include putative negative elements per2, per3, cry1, and cry2 and positive elements clock, bmal1, and bmal2 as well as ck1e. Furthermore, heterologous expression of positive element clock in COS7 cells has been shown to activate the chick AANAT expression and enzyme activity (62). In the present study retinal bmal1 and bmal2 are expressed rhythmically in LD and DD such that expression peaks at early night (ZT14/CT14), whereas clock does not appear rhythmically expressed. Of the putative negative elements in this tissue, only per3 and cry1 are expressed rhythmically, such that per3 is expressed predominantly in the late night/early day (ZT22-2) and late subjective night to early subjective day (CT22-6), whereas cry1 is expressed throughout the day in LD and subjective day in DD (Fig. 4A). The amplitudes of these rhythms are 2-3-fold. None of the other orthologs negative elements was found to be rhythmic, including per2 and cry2, nor was ck1e expressed rhythmically.
The temporal distributions of the mRNA for these clock proteins are interesting in several respects. First, there is no strict antiphase relationship of positive and negative elements that has been observed in Drosophila and mammalian models. Here, both bmals are expressed coincidentally with cry1, and of the rhythmic negative elements, cry1 and per3 are 180°in antiphase. This temporal pattern of clock gene mRNA abundance is similar but not identical to that found in the pineal gland (30). In the pineal gland, bmal1, bmal2, cry1, cry2, per2, and per3 are all expressed rhythmically with high amplitudes, and clock is expressed rhythmically in LD and DD, with a lower amplitude of 1.5-fold, all of which were confirmed by Northern analysis. The exception is the temporal distribution of retinal cry2, which is expressed bimodally in the pineal gland with peak mRNA values primarily during the late subjective day (CT10) and late subjective night (CT22). In the retina cry2 is not rhythmically expressed. Of course, we do not at this stage have any data concerning either the level or subcellular localization of the proteins that these genes encode. The similarities in temporal distribution between the chick retina and pineal gland and the differences between the temporal distributions of these mRNA species in these chick tissues and those of mammals and flies strongly suggest that other rhythmic mechanisms are in place. Furthermore, if clock gene expression regulates melatonin biosynthetic rhythms in these two tissues, one would predict that the downstream processes between clock function and its melatonin output differ between retina and pineal gland.
One might therefore ask, What are the other common elements between rhythmic retinal mRNA species in the present study and pineal rhythms in our previous work? First, in both tissues mRNAs encoding phototransduction and retinaldehyde transport are rhythmically expressed. This is not surprising, since both tissues are photoreceptive. Second, in both tissues, GAPDH and cytochrome c oxidase are expressed rhythmically such that GAPDH is expressed predominantly at CT10, and cytochrome oxidase is expressed highly from CT14 -18. Third, several ribosomal proteins and elongation factor 2 are expressed rhythmically in both tissues, suggesting protein synthesis and modification may play a role in regulating circadian function in these two tissues (Table I).
Several recent studies have pointed to rhythmic processes involving metabolic activity that are more or less independent of clock gene function. First, induction of clock gene rhythms does not necessarily induce metabolic rhythmicity in cultured cells (63). We have found that induction of robust clock gene rhythms in fibroblast lines by serum shock has no effect on glucose utilization in these cells even though rhythms of glucose utilization are accompanied by clock gene expression rhythms in SCN cells. Second, induction of metabolic rhythms in cultured astrocytes does not necessarily result in rhythmic clock gene expression, since glucose utilization rhythms can be induced in chick astrocytes with cycles of melatonin (64), but clock genes are not responsive to the hormone (65). Recent studies by McKnight and co-workers (66) have shown that the redox state in in vitro systems can determine the affinity of negative elements such as the cryptochromes for positive element activation of clock gene promoter regions. Both GAPDH and cytochrome oxidase have the potential of altering redox state via the reduction of NAD ϩ . Thus, it is possible that the rhythms in components of intermediary metabolic pathways we observe here in the retina and in the pineal gland elsewhere (30) may underlie a semi-independent oscillatory mechanism. Current research in our laboratory is focused on this issue.
It is interesting that several mRNA species involved in regulation of translation and protein trafficking are regulated on a circadian basis in the chick retina. Both 40 S and 60 S ribosomal proteins are regulated at the mRNA levels in this tissue in LD and DD as are several sequences associated with proteasomal proteolysis, suggesting a global regulation of many processes by the clock through the regulation of the translational machinery. This is a feature that is shared with the pineal gland as well (30). Although many researchers in the field have focused on rhythmic regulation of mRNA as a central mechanism in biological clock function (including the present study), translational control of clock components and their outputs that are independent of transcriptional regulation have been described in many model systems, including vertebrates. For example, in the filamentous fungus Neurospora crassa, circadian regulation of the clock gene frq and its protein FRQ can be separated by entrainment to long and short photoperiods, suggesting independent regulation of these two components (67). In Gonyaulax, circadian regulation of GAPDH levels and activity, a common clock-regulated protein (68), appears to be independent of mRNA levels (69), and whereas AANAT activity is rhythmically regulated in the sheep pineal gland, no rhythm in mRNA levels can be determined (54). This post-transcriptional regulation of key clock components and their outputs may be the result of circadian regulation of either rhythmic protein synthesis and/or proteolysis. Whether this post-transcriptional regulation is globally regulated or contains features that specifically target some protein regulation is not known at this stage.
In summary, the chick retina, an important component of the biological clock in birds, shares with the pineal gland many rhythmic mRNA species. These include components of the melatonin biosynthetic pathway as well as several clock genes. However, the details of their regulation are both similar and different, raising the reasonable possibility that the clocks in each of these tissues in the same organism are regulated dif-ferentially. Among the common elements between the rhythmic transcriptome in these tissues are key components of intermediary metabolism and protein synthesis and degradation, which should be considered candidates for clock regulation and its output.


extfieldsofstudy: Biology Medicine

text:
CpG-matured Murine Plasmacytoid Dendritic Cells Are Capable of In Vivo Priming of Functional CD8 T Cell Responses to Endogenous but Not Exogenous Antigens

Plasmacytoid dendritic cells (PDCs) are a unique leukocyte population capable of secreting high levels of type I interferon (IFN) in response to viruses and bacterial stimuli. In vitro experiments have shown that upon maturation, human and murine PDCs develop into potent immunostimulatory cells; however, their ability to prime an immune response in vivo remains to be addressed. We report that CpG-matured murine PDCs are capable of eliciting in naive mice antigen-specific CTLs against endogenous antigens as well as exogenous peptides, but not against an exogenous antigen. Type I IFN is not required for priming, as injection of CpG-matured PDCs into type I IFN receptor–deficient mice elicits functional CTL responses. Mature PDCs prime CTLs that secrete IFN-γ and protect mice from a tumor challenge. In contrast, immature PDCs are unable to prime antigen-specific CTLs. However, mice injected with immature PDCs are fully responsive to secondary antigenic challenges, suggesting that PDCs have not induced long-lasting tolerance via anergic or regulatory T cells. Our results underline the heterogeneity and plasticity of different antigen-presenting cells, and reveal an important role of mature PDCs in priming CD8 responses to endogenous antigens, in addition to their previously reported ability to modulate antiviral responses via type I IFN.

Introduction
DCs play a pivotal role in the control of innate and adaptive immune responses (1). They consist of a heterogeneous cell population, classified into distinct subsets according to surface phenotype, functional properties, and localization (2). In humans, an immature DC subset with plasmacytoid morphology (plasmacytoid DC [PDC]) represents a unique leukocyte population capable of secreting high levels of type I IFN in response to viruses and bacterial stimuli (3,4). It has been shown recently that human PDCs behave as bona fide DCs, as they efficiently prime naive antigen-specific CD8 T cells (5), and are capable of restimulating CD4 and CD8 responses upon influenza virus infection (6). In both experimental systems, CD4 and CD8 T cells expanded by PDCs were capable of IFN-␥ secretion. However, other investigators have shown that PDCs can differentiate allogeneic CD8 regulatory cells and Th2 responses (4,7), suggesting that PDCs may have a certain degree of plasticity in their ability to prime T cell responses.
Murine PDCs have been identified recently on the basis of high type I IFN secretion and their unique surface phenotype (CD11c dull , B220 ϩ , CD11b Ϫ , and Gr-1 ϩ ; references [8][9][10]. To date, all functional studies on murine PDCs have been performed in vitro using PDCs either isolated from spleen or differentiated from bone marrow precursors. It has been shown that freshly isolated murine PDCs express lower levels of MHC and costimulatory molecules than the myeloid CD11c high CD11b ϩ subset (myeloid DC [MDC]), possibly accounting for their reported poor stimulatory capacity for allogeneic and naive T cells (8)(9)(10)(11). In contrast, PDCs matured with viral or CpG stimulation are potent The online version of this article includes supplemental material.
In Vivo Priming by Murine PDCs 568 APCs, capable of stimulating proliferation of allogeneic T cells and naive transgenic CD4 and CD8 T cells (8,10,(12)(13)(14). Depending on antigen dose and Toll-like receptor engagement, murine PDCs show flexibility in their T cell polarizing capacity, generally eliciting Th1 responses at high and Th2 responses at low antigen doses (15). In addition, immature PDCs have been shown to differentiate T regulatory cells, capable of suppressing antigen-specific T cell proliferation (13,16,17).
Although in vitro experiments indicate that mature PDCs are potent immunostimulatory cells, it remains unclear whether they can prime antigen-specific immune responses in vivo in naive nontransgenic animals. To address this question, we set up an in vivo priming model in which we monitored ex vivo by tetramer analysis the proliferation of antigen-specific T cells after injection of PDCs either freshly isolated from the spleen or from FLT3 ligand (FLT3-L)supplemented murine bone marrow cultures (18). We report that CpG-matured PDCs prime CTLs specific for endogenous but not exogenous antigens. CTLs primed by PDCs acquire potent in vivo cytolytic activity, are capable of IFN-␥ secretion upon peptide stimulation, and protect mice from a subsequent tumor challenge. Priming is dependent on direct presentation of the antigen by the injected DCs and does not require responsiveness to type I IFN. Conversely, immature PDCs do not induce proliferation of antigen-specific CTLs in vivo. However, in contrast to what was observed in vitro, administration of immature PDCs does not prevent responses to subsequent challenges with viruses or DCs expressing the relevant antigen.
Generation of Bone Marrow-derived DCs. Culture medium was RPMI 1640 supplemented with 2 mM l -glutamine, 1% nonessential amino acids, 1% pyruvate, 50 g/ml kanamycin, 0.05 mM 2-mercaptoethanol (GIBCO BRL), and 10% FCS (Hyclone). Bone marrow cells were isolated by flushing femurs and tibia with complete medium. RBCs were lysed with RBC lysis solution (Puregene; Gentra Systems). The recovered cells were plated in culture medium containing 100 ng/ml FLT3-L (R&D Systems) at 10 6 cells/ml in six-well plates in a volume of 5 ml as described previously (18). Every 3-4 d, 2.5 ml of medium was replaced with fresh medium and FLT3-L. At day 10, half of the cultures were matured with 5 g/ml phosphorothioate CpG DNA 1826 (Coley Pharmaceutical). In some experiments, bone marrow cultures were incubated with 500 g/ml of soluble ovalbumin (fraction VII; Sigma-Aldrich) for 20 h before sorting (CpG was added to part of the cultures 4 h after ovalbumin). At day 11, the cultures were phenotyped by FACS ® analysis with the following mAbs: CD11b-FITC, B220-PE, and CD19-APC (all obtained from BD Biosciences). PDCs (CD11b Ϫ B220 ϩ CD19 Ϫ ) and MDCs (CD11b ϩ B220 Ϫ CD19 Ϫ ) were isolated by a combination of magnetic (MACS; Miltenyi Biotec) and cell sorting (Moflo; DakoCytomation). The purity was always Ͼ 97%, and CD11b ϩ cells were not detected upon reanalysis of PDC preparations.
Immunization Protocols. Sorted cells were left unpulsed or pulsed with 1 g/ml of peptide in serum-free medium for 2 h at 37 Њ C, extensively washed, and diluted in PBS. 200 l of cells was injected into the lateral tail vein. Animals were boosted by i.v. injection of 3 ϫ 10 5 BM-DCs or 10 6 PFUs of UV-inactivated recombinant vaccinia encoding the UTY 246-254 or SMCY 738-746 minigenes or the full length ovalbumin protein (22).
Generation of Recombinant Vaccinia Viruses. Recombinant vaccinia viruses (WR strains) encoding the UTY 246-254 and SMCY 738-746 minigenes were made by cloning each insert into the thymidine kinase gene using the vector pSC11 as described previously (23).
Isolation of PBLs and Tetramer Staining. Blood was taken from the tail vein, and PBLs were isolated after depletion of RBCs with RBC lysis solution (Puregene; Gentra Systems). Cells were resuspended in 25 l of complete medium and incubated with 0.5 g of tetramer for 25 min at 37 Њ C. Cells were washed and incubated with rat anti-mouse CD8 ␣ (BD Biosciences) for 20 min at 4 Њ C. Cells were washed twice and analyzed using a FACSCali-bur™ with CELLQuest™ software.
In Vivo Killing Assay. To assess cytotoxicity, immunized and control mice were injected with a mixture of four differentially labeled syngeneic splenocyte populations, loaded or not with 10 g/ml UTY 246-254 peptide; three populations were labeled with different concentrations of carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes) and one with 10 M of the dye 5-(and 6)-([{4-chloromethyl}benzoyl]amino) tetramethylrhodamine (Molecular Probes; references 24,25). Labeled cells were pooled and injected at 10 7 cells/mouse into the tail vein. Cytotoxicity was assessed by FACS ® analysis on blood taken from the lateral tail vein at different time points. The mean percentage lysis of peptide-loaded target cells was calculated relative to antigennegative controls with the following formula: 100 Ϫ (100 ϫ adjusted percent survival). Adjusted percent survival was calculated as follows: (percent survival Ag ϩ /percent survival Ag Ϫ )/mean percent survival in control animals.
ELISPOT. Blood was taken from the tail vein, and PBLs were isolated after depletion of RBCs with RBC lysis solution (Puregene; Gentra Systems). In some experiments, RBCdepleted splenocytes were used as responders. Analysis of IFN-␥ production in response to stimulation with 10 M peptide for 16 h 569 was performed on MultiScreen-IP high protein-binding 96-well plates (Millipore) using MabTech mouse IFN-␥ ELISPOT kit according to the manufacturer's instructions. In all experiments, stimulation with 1 g/ml PHA served as positive control.
Tumor Immunity Assay. 10 d after priming, mice were challenged with subcutaneous injection of 10 6 B16-F10 tumor cells expressing the LCMV gp 33-41 minigene (26). Mice were monitored for tumor growth every 3-4 d, and the tumor size for each group was calculated as the mean of the products of bisecting diameters ( Ϯ SEM). Measurements were terminated for each group when the first animal developed ascitis, when the tumor became ulcerated, or when it grew in excess of 200 mm 2 .
Intracellular Cytokine Staining. Spleens were harvested 8-9 d after priming, and lymphocytes were isolated after depletion of RBCs with RBC lysis solution (Puregene; Gentra Systems). Cells were plated in complete medium and stimulated with 10 g/ml UTY 246-254 peptide, 10 Ϫ 6 M PMA (Sigma-Aldrich), and 1 g/ml ionomycin (Sigma-Aldrich) or left unstimulated. 5 g/ml Brefeldin A (Sigma-Aldrich) was added after 1 h, and cells were collected after a total of 6 h. Cells were fixed in 2% paraformaldehyde, permeabilized in saponin buffer (27), and stained with antibodies to mouse IFN-␥ -FITC, IL-2-PE, IL-10-PE, and IL-4-APC (BD Biosciences). Tetramer staining was performed on a sample of unstimulated splenocytes as described in previous paragraphs for blood PBLs.

Intravenous Injection of CpG-matured Male PDCs Induces
Functional CTL Responses. Thus far, the stimulatory capacity of murine PDCs has been investigated only in vitro against transgenic T cells (8,10,(12)(13)(14). Therefore, we de-veloped an in vivo model to study the ability of PDCs to prime and polarize antigen-specific T cells from naive nontransgenic precursors. We differentiated both PDCs and control MDCs from murine bone marrow cultures supplemented with FLT3-L (18) and isolated the two DC populations by a combination of magnetic and cell sorting. In preliminary experiments, we observed that MDCs isolated from FLT3-L or GM-CSF ϩ IL-4-supplemented cultures elicited qualitatively and quantitatively comparable CTL responses (unpublished data) and, therefore, restricted our subsequent analysis to FLT3-L-differentiated MDCs. The phenotype of bone marrow-derived PDCs and MDCs is shown in Fig. S1 (available at http://www.jem.org/cgi/ content/full/jem.20031059/DC1).
In a first set of experiments, we investigated the response to an endogenous antigen, the male-specific transplantation antigen H-Y. When injected with male cells, female mice of the H-2 b haplotype develop an immunodominant H-2-D brestricted response against the UTY 246-254 peptide (WMHH-NMDLI) that can be monitored by tetramer analysis (28,29).
UTY 246-254 -specific CTL responses were monitored ex vivo in the blood of female C57BL/6 mice injected with male PDCs or MDCs, either immature or CpG matured. Mature PDCs were as efficient as mature MDCs in eliciting UTY 246-254 -specific CTLs, as detected by tetramers ( Fig. 1  A). In contrast, although immature MDCs always elicited a distinct CTL population (although lower in numbers as compared with mature MDCs), immature PDCs never induced proliferation of UTY 246-254 -specific CTLs above the detection limit of the tetramer staining ( Fig. 1 A). Similar results were obtained when responses to the subdominant H-2-D b -restricted SMCY 738-746 epitope (28) were monitored (unpublished data; Fig. S2, available at http:// www.jem.org/cgi/content/full/jem.20031059/DC1).
To better evaluate quantitatively the CTL responses, we titrated the numbers of DCs. At low numbers (3,000 cells/ mouse), MDCs were far more efficient than PDCs in eliciting UTY 246-254 -specific CTLs ( Fig. 1 B), whereas at 10,000 cells/mouse, PDCs and MDCs elicited a similar The functional state of the induced UTY 246-254 -specific CTLs was investigated by assessing their cytotoxic capacity and cytokine secretion upon antigen exposure. Cytotoxicity was assayed in vivo against syngeneic splenocyte targets, either male or peptide-pulsed female that had been labeled with a fluorescent dye and injected into the lateral vein 10 d after priming. Specific lysis of the antigen-expressing cells was determined against control female splenocytes not pulsed with the UTY 246-254 peptide. UTY 246-254 -specific CTLs primed by mature PDCs efficiently lysed pulsed with 10 g/ml of peptide female cells, as well as unpulsed male splenocytes (Fig. 2, B and C). Lysis of unpulsed male splenocytes, expressing a much lower density of UTY 246-254 -D b complexes than UTY 246-254 peptide-pulsed cells, is indicative of expansion of high affinity CTLs. The majority of peptide-pulsed female targets were lysed within the first 17 h, whereas lysis of unpulsed male cells continued over the next 96 h, consistent with UTY 246-254 -D b complexes being presented at lower density but continuously over time (Fig. 2 B; not depicted). The cytolytic activity detected in mice primed by immature and mature MDCs correlated with the extent of CTL priming. No specific lysis above background was observed in mice primed by immature PDCs, mirroring the lack of detectable CTLs in the blood.
ELISPOT assays were performed to assess the capacity of UTY 246-254 -specific CTLs to secrete IFN-␥ in response to peptide stimulation. As shown in Fig. 3, and in agreement with the tetramer data, mice primed by either mature PDCs or immature and mature MDCs were capable of recognizing the UTY 246-254 peptide in an ex vivo assay, without further in vitro restimulation. No specific IFN-␥ secretion was observed in mice primed by immature PDCs.
Intravenous Injection of CpG-matured Male PDCs Induces Type I Polarization of Splenic UTY 246-254 -specific CTLs. To further characterize the type of polarization induced by priming with mature PDCs, freshly isolated splenocytes  . Intravenous injection of CpG-matured male PDCs induces IFN-␥-secreting CTL. C57BL/6 mice (n ϭ 5) were primed as described in Fig. 1. ELISPOT assay was performed on blood PBLs to assess IFN-␥ secretion by antigen-specific cells in response to 10 g/ml UTY 246-254 peptide 7 d after priming. All animals showed comparable responses to PHA stimulation (not depicted).
were stimulated in vitro with the UTY 246-254 peptide or PMA ϩ ionomycin 8 d after priming. Analysis of intracellular cytokines revealed that CD8 T cells secreted the type 1 cytokine IFN-␥ in response to both the specific peptide and PMA ϩ ionomycin (Fig. 4). IL-2 was also detected in CD8 T cells stimulated with PMA ϩ ionomycin. In contrast, CD8 cells did not secrete the type 2 cytokines IL-4 and IL-10 in response to either stimulation protocol. IL-4 and IL-10 were secreted only by the CD8 negative cells in response to PMA ϩ ionomycin. A similar pattern of polarization was observed in mice primed by mature MDCs (unpublished data). Although the frequencies of tetramer ϩ cells in the spleen and in the blood were comparable, a higher proportion of splenic UTY 246-254 -specific CTLs secreted IFN-␥ in response to the specific peptide ( Fig. 4; not depicted), which may reflect homing of effector T cells to the spleen. We conclude that mature PDCs are able to efficiently prime antigen-specific CTLs, capable of cytolytic activity and IFN-␥ secretion.
Priming of UTY 246-254 -specific CTLs by Freshly Isolated CpG-matured Splenic PDCs. It has been shown previously that freshly isolated splenic PDCs are less mature than their bone marrow-derived counterparts, hence they are also less immunostimulatory (15). Preliminary experiments showed that injection of immature splenic MDCs, in contrast to bone marrow-derived MDCs, did not elicit UTY 246-254 -specific CTLs detectable by ex vivo tetramer staining (unpublished data). Therefore, we isolated splenic PDCs and MDCs from male mice previously injected with CpG to induce in vivo DC maturation. Both MDCs and PDCs elicited UTY 246-254 -specific CTLs, although responses were much weaker than those elicited by equal numbers of bone marrow-derived DCs and were detectable only upon in vivo restimulation with vaccinia virus Figure 4. Intravenous injection of CpG-matured male PDCs induces type I polarization of spleen CTL. C57BL/6 mice (n ϭ 3) were injected i.v. with 10 5 male CpG-matured MDC or PDCs. Splenocytes from a mouse primed by CpG-matured PDCs were in vitro restimulated with UTY 246-254 peptide or PMA and ionomycin, and intracellular cytokine accumulation was assessed as described in Materials and Methods 8 d after priming. The tetramer staining for the same sample is also shown. One experiment representative of three is shown. Similar profiles were obtained in mice primed by CpG-matured male MDC, whereas naive female C57BL/6 controls did not respond to the peptide. encoding the UTY 246-254 minigene (Fig. 5 A). UTY 246-254specific CTLs primed by mature splenic PDCs and MDCs were functional, as shown by IFN-␥ secretion in response to the cognate peptide in an ex vivo ELISPOT assay (Fig. 5 B).
Priming of UTY 246-254 -specific CTLs Relies upon Direct Presentation of the Male Antigen by the Injected DCs. Priming of UTY 246-254 -specific CTLs by CpG-matured PDCs and MDCs could be due to direct presentation of the UTY 246-254 peptide by the male APCs. Alternatively, proliferation of UTY 246-254 -specific CTLs could be due to uptake and presentation of male APC debris by resident female DCs, a mechanism known as cross-priming. Indeed, cross-priming has been shown to be effective for the generation of cytotoxic T cells, and it may be the dominant route for priming of some responses (30,31). In vitro experiments showed that presentation of the UTY 246-254 epitope is entirely TAP-dependent as UTY 246-254 -specific T cells did not recognize TAP-1-deficient male APCs (unpublished data). Therefore, we used TAP-1-deficient male BM-DCs as immunogens to distinguish between direct versus crosspresentation. Injection of as many as 10 6 male TAP-1 Ϫ/Ϫ BM-DCs, either immature or CpG matured, failed to prime UTY 246-254 -specific CTLs in female C57BL/6 mice ( Fig. 6). In contrast, control mice developed good responses after injection with 10 5 wild-type BM-DCs. After boosting with vaccinia-UTY 246-254 minigene, only mice that had been primed with 10 6 male TAP-1 Ϫ/Ϫ DC (i.e., 10 times more APCs than used in previous experiments) showed enhanced CTL responses (unpublished data). In agreement with the observation that UTY 246-254 -specific CTL responses cannot be efficiently generated upon priming by ␤-2m-deficient APCs (28), we conclude that the role of cross-priming in generating UTY 246-254 -specific CTLs in this system is marginal, and can only be appreciated when animals are injected with large numbers of APCs. Therefore, proliferation of UTY 246-254 -specific CTLs in our in vivo model can be accounted for by direct presentation of the endogenous antigen by the injected PDCs and MDCs.

Lack of Priming by Intravenous Injection of Immature Male PDCs Does Not Prevent Subsequent Induction of Functional
UTY 246-254 -specific Responses. The inability of immature PDCs to prime antigen-specific CTLs could reflect the lack of expression of costimulatory or adhesion molecules essential to trigger naive T cell proliferation. Alternatively, immature PDCs could have induced the proliferation of anergic or regulatory cells nonreactive to further antigenic challenge or unable to bind UTY 246-254 -D b tetramers (32,33). The possibility of anergy or negative regulation was ruled out by demonstrating that mice injected previously with immature PDCs developed cytolytic male-specific CD8 T cells upon boosting by splenocytes (Fig. 7, A and  B). The acquisition of cytolytic activity correlated with the appearance of tetramer ϩ CTLs in the blood (Fig. 7 C), which also secreted IFN-␥ in response to the cognate peptide in an ex vivo ELISPOT assay (Fig. 7 D).
As a further assessment of functional activity, we showed that previous injection of immature PDCs did not prevent expansion of UTY 246-254 -specific CTLs 1 wk after in vivo boosting with either male BM-DCs or vaccinia virus encoding the UTY 246-254 minigene (Fig. 7, E and F).
We conclude that, despite the inability to detect UTY 246-254 -specific CTLs after one round of injection with immature PDCs (by either tetramer, ELISPOT, or in vivo killing assays), there was no evidence of tolerance to subsequent antigenic challenge.
Type I IFN Receptor-deficient CpG-matured Male PDCs Prime IFN-␥ Secreting UTY 246-254 -specific CTLs. PDCs are a unique leukocyte population capable of secreting high levels of type I IFN in response to viruses and bacterial stimuli (8)(9)(10). Because type I IFN is important for the survival and proliferation of memory CD8 T cells (34,35), we investigated the priming capacity of CpG-matured PDCs in mice lacking type I IFN receptor (129A mice) (19).
As compared with 129 wild-type mice, 129A mice had normal numbers of PDCs ex vivo in the spleen and in vitro after culturing bone marrow cells in the presence of FLT3-L (unpublished data). In addition, 129A PDCs underwent maturation after CpG treatment, although to a lower extent than their wild-type counterpart (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20031059/DC1). Female 129A mice injected with male CpG-matured 129A PDCs developed antigen-specific CTLs, which could also be boosted by UV-inactivated vaccinia UTY 246-254 minigene, as shown previously for C57BL/6 mice (Fig. 8 A). In addition, UTY 246-254 -specific CTLs primed by mature 129A PDCs secreted IFN-␥ in response to the cognate peptide in an ex vivo ELISPOT assay (Fig. 8 B). These results suggest that in the absence of viral infection, type I IFN responsiveness is not essential for CTL priming by CpG-matured PDCs. Priming by CpG-matured PDCs Induces Protective CTL Responses. We have shown that PDCs can efficiently present an endogenous antigen and prime naive CTL precursor. We extended these results by analyzing the ability of peptide-pulsed PDCs to prime antigen-specific CTLs. We pulsed PDCs and MDCs with the LCMV gp 34-41 peptide (K b restricted) and monitored ex vivo LCMV gp34specific CTLs by tetramer staining. In accordance with what was observed previously, CpG-matured PDCs and MDCs induced similar proliferations of LCMV gp34-CTL; immature MDCs were somewhat intermediate, and no priming was induced by immature PDCs (Fig. 9 A).
These animals were challenged at day 10 with B16-F10-gp33, a derivative of the aggressive melanoma B16 that grows rapidly when injected in C57BL/6 mice (26). As shown in Fig. 9 B, mice that had been primed by CpGmatured PDCs or MDCs were completely protected from tumor growth and remained tumor free for up to 2 mo (not depicted). Mice primed by immature MDCs succumbed to the tumors, although they did so 2 wk later than mice injected with immature PDCs (which did not develop LCMV gp34-CTL) or unimmunized control groups.
Lack of Presentation of Exogenous Antigens by Bone Marrowderived PDCs. To further characterize the antigen-processing capacity of PDCs as compared with classical MDCs, we investigated the presentation of a model exogenous antigen, soluble ovalbumin. We sorted PDCs and MDCs from bone marrow cultures prepulsed with ovalbumin in the presence or absence of a maturation stimulus to induce cross-presentation (36). Ex vivo SIINFEKL-K b tetramer staining detected primary CTL responses only upon injection of mature MDCs (unpublished data). After boosting with vaccinia virus encoding full-length ovalbumin, mice primed by ovalbumin-loaded PDCs did not expand SIIN-FEKL-specific CTLs above controls, regardless of the PDC maturation stimulus (Fig. 10 A). In contrast, CTL responses primed by MDCs were boosted two-to fourfold.
As a control, both CpG-matured PDCs and MDCs pulsed with the OVA 257-264 peptide elicited functional SI-INFEKL-specific CTLs detectable in the blood by tetramer staining (Fig. S4, available at http://www.jem.org/cgi/  content/full/jem.20031059/DC1). Mice primed by MDC showed significant IFN-␥ secretion in response to ovalbumin class I and class II peptides, whereas the response in mice primed by PDCs was comparable to that of control mice infected with vaccinia-OVA only (Fig. 10 B).

Discussion
In this paper, we have shown that CpG-matured murine PDCs efficiently prime antigen-specific CTLs in vivo from naive nontransgenic precursors. PDC-primed CTLs display cytolytic activity in vivo against target cells expressing the relevant antigen and are capable of protecting mice from a subsequent challenge with a tumor recombinant for the immunizing peptide. CTL priming occurs after injection of peptide-pulsed PDCs, but more importantly, we dem-onstrated that PDCs present endogenous antigens for which the density of MHC-peptide complexes at the cell surface is much lower than after peptide pulsing. However, PDCs are not able to present exogenous antigens, in contrast to MDCs, thus underlining the heterogeneity within the DC populations.
Previous works showed that, upon in vitro maturation, PDCs develop into potent immunostimulatory cells, although not to the same extent as control MDCs (8,10,(12)(13)(14). In our in vivo experiments, mature PDCs and MDCs injected at high numbers elicited comparable responses, qualitatively and quantitatively, whether presenting the endogenous antigen or peptide pulsed. Only when limiting numbers of DCs were injected, MDCs were more stimulatory than PDCs. In line with our results, Dalod et al. (37) showed recently that PDCs purified after in vivo challenge with murine cytomegalovirus (MCMV) became as potent as other DC subsets for activation of naive CD8 T cells. Dalod et al. did not address whether PDCs could also present endogenous viral antigens because MCMV did not infect PDCs, but only induced their maturation. Our results showing priming of UTY 246-254 -specific CTL responses by male PDCs strongly suggest that PDCs, infected by viruses inducing their maturation, would be capable of priming virus-specific T cell responses.
In contrast with the majority of the analyses, Krug et al. have shown that splenic PDCs failed to stimulate a strong proliferation of naive CD4 and CD8 T cells, even after in vivo viral stimulation, questioning whether PDCs belong to the DC system (11). Conversely, we have shown that, although less efficient than bone marrow-derived PDCs, freshly isolated in vivo-matured splenic PDCs are able to prime UTY 246-254 -specific CTLs. It is possible that maturation induced by either CpG (our model) and MCMV (37) or VSV (11) may not generate functionally equivalent cells as a consequence of different Toll-like receptor engagement and, therefore, account for the observed discrepancies. With respect to the model antigen used, we analyzed ex vivo CTL responses specific for the immunodominant H-Y UTY 246-254 -H-2-D b epitope and the subdominant SMCY 738-746 -H-2-D b epitope in nontransgenic animals. In contrast, Krug et al. have looked at the endogenous presentation of the SMCY 738-746 -H-2-D b epitope using TCR transgenic mice (38). These mice fail to reject male skin grafts, and SMCY 738-746 TCR transgenic CD8 T cells do not induce graft versus host disease when transferred in male nude mice (39,40), suggesting that this receptor may have poor reactivity for its ligand. In addition, SMCY 738-746 TCR transgenic CD8 T cells have a defect in cytotoxicity in response to male targets that can be overcome at elevated densities of the cognate peptide (41,42), which may be present on the surface of MDCs but not PDCs (11).
We have not studied the capacity of mature bone marrow-derived PDCs to present endogenous antigens to CD4 T cells. However, as the development of the UTY 246-254specific CTL response is strictly dependent on T cell help from H-Y-A b CD4 cells (28), we speculate that ma- ture PDCs might have primed CD4 as well as CD8 malespecific cells.
It has been shown that human immature MDCs induce differentiation of CD4 and CD8 regulatory T cells, both in vivo and in vitro (43,44). In the mouse, targeting of antigen in vivo to immature DCs induces CD4 ϩ CD25 ϩ regulatory T cells and CD8 T cell tolerance (45,46). Upon injection of immature MDCs, we elicited functional CTLs, although in lower numbers than with mature MDCs, and this can be explained by the fact that these cells may have undergone a limited degree of maturation during the culture/sorting period (unpublished data). Regulatory T cells have also been differentiated in vitro by CD40L-activated human PDCs (7) and murine immature PDCs (13,16,17), and it has been hypothesized that thymic PDCs might be involved in T reg differentiation (13). However, in our in vivo priming model, we did not elicit any antigen-specific CTLs when mice were injected with immature PDCs (male DCs or peptide pulsed), making it difficult to test for any regulatory activity. Nevertheless, exposure to immature PDCs did not prevent induction of immunity after subsequent challenge with DCs, recombinant virus, or splenocytes, ruling out establishment of long-term tolerance. Although these results might at first seem difficult to reconcile with published data, they may be explained by the different experimental systems used. Indeed, previous in vivo studies have been performed with adoptively transferred naive transgenic T cells, whereas our paper relies on endogenous CTL precursors present in much lower numbers. The development of antibodies to specifically target antigens to PDCs in vivo, as described for CD8␣ ϩ DCs via DEC-205 (45,46), will help to address whether immature PDCs may also induce tolerance and T regulatory cells.
With respect to T cell polarization, we have shown that CTLs primed by CpG-matured PDCs secreted mainly IFN-␥ in response to the cognate antigen both by ex vivo ELISPOT and intracellular cytokine assays. Other investigators have reported high IFN-␥ by transgenic CTLs polarized in vitro by mature PDCs pulsed with OVA 257-264 or LCMV gp [33][34][35][36][37][38][39][40][41] peptides (12,37). A flexibility of murine PDCs in directing Th1 and Th2 development of CD4 transgenic T cells has been described, depending on antigen dose and Toll-like receptor engagement (15). This is likely to reflect differences in polarization of CD4 and CD8 cells, consistent with the lack of secretion of IL-4 by PMA ϩ ionomycinstimulated CD8 splenocytes (Fig. 3). We have not been able to induce UTY 246-254 -specific CTL proliferation when mice were injected with immature PDCs (male PDCs or peptide pulsed); therefore, we cannot comment on the CTL polarizing capacity of PDCs at different stages of maturation.
We have shown that, in the absence of viral infection, type I IFN is not required for priming because injection of CpG-matured PDCs into type I IFN receptor-deficient mice (129A) elicits functional CTL responses. In contrast, during a viral infection, the importance of PDC-derived type I IFN in initiating innate immune responses and in the cross-talk with other DC subsets for induction of adaptive immunity has been very elegantly demonstrated (37,47). However, we cannot exclude the possibility that other recently described members of the IFN family may play a key role in PDC stimulatory capacity (48,49), and some redundancy in the IFN system may be envisaged. Such redundancy may be implied by the observation that 129A mice have PDCs (Fig. S3, available at http://www. jem.org/cgi/content/full/jem.20031059/DC1) in contrast to what was reported for IFN consensus sequence-binding protein (a transcription factor involved in the IFN signaling pathway)-deficient mice that lack PDCs and have a defect in the activation of CD8␣ ϩ DCs (50)(51)(52). In addition, 129A PDCs mature in response to CpG treatment, although to a lower extent than their wild-type counterpart, consistent which was with what was reported previously (53)(54)(55). This level of maturation is indeed sufficient for in vivo priming of functional CTL responses, although in other experimental models, amplification of DC maturation by IFN signaling may be required for optimal regulation of the immune response (37).
Although in vivo mature PDCs can induce proliferation of functional CTLs specific for an endogenous antigen as well as for exogenously loaded peptides, they are unable to present exogenous antigens, even in the presence of maturation signals known to activate cross-presentation in MDCs (36). This observation is consistent with a recent paper showing that PDCs were far less efficient compared with other DC subsets in presenting s.c. injected Leishmania major antigens (56). Lack of presentation of exogenous antigens could be due to poor endocytic activity compared with MDCs (unpublished data; references 57, 58), or to differences in the antigen-processing and -presenting machinery between MDCs and PDCs, consistent with the different expression pattern of cathepsins in human MDCs and PDCs (59). Our results underline the heterogeneity and plasticity within the APC family, highlighted previously between CD8␣ ϩ and CD8␣ Ϫ MDCs (60)(61)(62)(63). We envision that in vivo PDCs may contribute to priming antigen-specific CD8 and CD4 T cell responses by efficiently presenting endogenous antigens, whereas their role in priming T cell responses to exogenous antigens would be negligible; in this respect, differing from both CD8␣ ϩ and CD8␣ Ϫ MDCs. In addition, PDCs could modulate the function of other DC subsets by inducing their functional maturation and promoting cross-presentation via type I IFN secretion (64). Finally, these data validate in vivo our previous results on the ability of human PDCs to prime antigen-specific naive T cells (5), and provide a rationale for the combined use of mature MDCs and PDCs in vaccine trials to optimize the induction of innate and adaptive immune responses.
We would like to thank Drs. A. O'Garra for helpful discussion and C. Hetherington for care of the animal colonies.
This work was funded by Cancer Research (project grant C399/ A2291) and the Cancer Research Institute.