Modulation of T cell development and activation by novel members of the Schlafen (slfn) gene family harbouring an RNA helicase-like motif

Peter Geserick, Frank Kaiser, Uwe Klemm, Stefan H. E. Kaufmann and Jens Zerrahn

Department of Immunology, Max-Planck Institute for Infection Biology, Schumannstr. 21–22, 10117 Berlin, Germany

Correspondence to: J. Zerrahn; E-mail: zerrahn{at}mpiib-berlin.mpg.de


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The regulatory networks governing development and differentiation of hematopoietic cells are incompletely understood. Members of the Schlafen (Slfn) protein family have been implicated in the regulation of cell growth and T cell development. We have identified and chromosomally mapped four new members, slfn5, slfn8, slfn9 and slfn10, which belong to a distinct subgroup within this gene family. The characteristic feature of these proteins is the presence of sequence motifs identifying them as distinct members of the superfamily I of DNA/RNA helicases. A significant role of these newly identified members in hematopoietic cell differentiation is suggested based on their differential regulation (i) in developing and activated T cells, (ii) in LPS or IFN{gamma} activated macrophages, (iii) upon IL6 or LIF driven terminal differentiation of myeloblastic M1 cells into macrophage-like cells, and (iv) in splenocytes of mice infected with Listeria monocytogenes. In contrast to wild-type cells, IRF-1 and IFN{alpha}/ßR deficient macrophages, although undergoing growth arrest, fail to upregulate slfn gene expression upon IFN{gamma} or LPS stimulation, respectively. Therefore, an essential participation in IFN{gamma} or LPS induced growth arrest appears unlikely. Likewise, ectopic expression of the newly identified slfn family members in fibroblasts did not reveal a general impact on growth control. In contrast, transgenic T-cell specific expression of a representative member of this new subfamily, slfn8, resulted in profoundly impaired T cell development and peripheral T cells showed a reduced proliferative potential. Thus, functional participation of slfn8 in the regulatory networks governing T cell development and growth appears to be cell type specific.

Keywords: cellular differentiation, gene regulation, macrophages, thymus, T lymphocytes


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cellular differentiation is a process of gradual acquisition of cell type specific properties governed by extracellular factors and cell–cell interactions. In the hematopoietic system, differentiating cells, like developing T and B cells, transit through distinct phenotypic and molecular stages (13). The homeostasis of mature lymphoid and myeloid cells is in part controlled by intrinsically hard-wired genetic programs (47). In addition, homeostatic control is further tuned by extrinsic signals like cytokines, which e.g. influence longevity of memory T cells (8,9). In case of mature naive lymphocytes, further differentiation is induced by antigen specific activation during an ensuing immune response. In principal, differentiation is tightly interconnected with the cell cycle control machinery (10,11). This is instructively illustrated by developing and mature T cells. Thymocytes traverse distinct proliferative stages (12), seed as resting mature cells in the periphery, expand upon antigen specific encounter, subsequently undergo apoptosis or convert to long-lived memory cells which can re-expand upon re-encounter of antigen (13). Unravelling the participating regulatory networks is crucial for an in-depth understanding of hematopoietic cell homeostasis and disease.

Members of the recently identified Schlafen (slfn) gene family have been implicated in an as yet unidentified regulatory mechanism involved in T-cell development and growth control (14). The prototype of this family, slfn1, has been identified by subtractive hybridization as a gene which is highly upregulated in positively selected, mature CD4 and CD8 thymocytes as compared to immature progenitors. Likewise, peripheral resting T cells express abundant slfn1 mRNA levels, which markedly decrease upon T cell receptor (TCR) triggered activation. Transgenic slfn1 expression perturbs thymocyte development, evoking a severe block at the CD25+CD44 stage within the immature CD4CD8CD3 (DN) progenitor population. Accordingly, ectopic expression in fibroblasts causes arrest in the G1 phase of the cell cycle (14). Thus, slfn1 seems to be a negative regulator of cell growth, because its natural or ectopic expression correlates with resting cellular states. The exact molecular functions of Slfn proteins are unknown and revelation is hampered by the lack of homology to any functionally characterized protein family.

We report here the identification of a new, distinct subgroup within the slfn gene family. These genes are differentially regulated in various hematopoietic cells, like developing or activated peripheral T cells, in macrophages upon activation or during myeloid terminal differentiation. A characteristic feature of these new members is an additional C-terminal domain, not present in the previously described family members, with motifs homologous to the sequential, canonical fingerprints that are characteristic for DNA/RNA helicases belonging to the superfamily I. Analysis of slfn8 transgenic mice suggests an important regulatory role of slfn 8, and inferentially of the other new Slfn family members in the development and differentiation of T cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
TNFR1–/– mice (15), IRF-1–/– (16), IFN{gamma}R–/– (17) and IFN{alpha}/ßR–/– mice (18) were kindly provided by Dr Klaus Pfeffer (University of Düsseldorf, Düsseldorf, Germany), Dr T. Mak (Amgen, Toronto, Canada), Dr Michel Aguet (Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland) and Dr R. M. Zinkernagel (Institute for Experimental Immunology, Zürich, Switzerland), respectively. C57BL/6 (B6), 129/SvJ (129) and B6 RAG1–/– mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were maintained under specific pathogen-free conditions at our animal facilities at the Federal Institute for Risk Assessment (Berlin). Mice were infected i.v. with 5 x 103 Listeria monocytogenes EGD.

Identification of new slfn gene family members
Analysis of a differential cDNA library (19) generated by suppression-subtractive hybridization from T and B cell depleted splenocytes from L. monocytogenes-infected mice 2 days post infection, with reference to cells from uninfected animals, revealed presence of an incomplete cDNA being partially homologous to the known slfn genes. A genomic cosmid library derived from 129/Ola mice (RZPD number 121) was obtained from the Resource Center and Primary Database (RZPD, Berlin, Germany) and screened by PCR with various primers designed according to the sequence of the newly identified slfn gene cDNA. The identified PCR positive, LAWRIST7 based cosmids P06130Q2, D2074Q1, E12428Q2 and P09246Q2 were further analyzed by sequencing either directly or upon subcloning of EcoRI fragments. This led to the identification of four new members of the slfn gene family localized on the overlapping cosmids. The acquired sequence information was used for 5'-RACE analysis employing SMARTTM RACE cDNA amplification (Clontech, Palo Alto, CA), performed according to the recommendations of the manufacturer, identifying the genomic locations of non-coding exons. Full-length cDNAs were subsequently obtained by RT–PCR with Pfu-Polymerase (Stratagene) using cDNA from IFN{gamma}-stimulated bone marrow-derived macrophages (BMM) from B6 and 129 mice, cloned and used as PCR templates for the generation of end modified cDNA variants suited for cloning into appropriate expression vectors. Accession numbers are: slfn5 (B6), AY261804; slfn8 (B6), AY261798; slfn9 (B6), AY261800; slfn10 (B6), AY261802; slfn5 (129), AY261805; slfn8 (129), AY261799; slfn9 (129), AY261801; slfn10 (129), AY261803.

Cells
BMM from B6, IRF-1–/– and IFN{alpha}/ßR–/– mice were prepared as described elsewhere (20) and the cells were used at day 7–9 for all experiments. The BMM were cultured in DMEM (Biochrom KG, Berlin, Germany) supplemented with 10% heat-inactivated FCS, 5% horse serum, 20% L-cell conditioned medium, 5 x 10–5 M 2-ME, 20 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate and antibiotics. Reagents used for stimulation were: 500 ng/ml LPS from E. coli J5-Rc mutant (Sigma) or 500 U/ml recombinant murine IFN{gamma} (kindly provided by Dr G. Adolf, Boehringer Ingelheim, Austria). To analyze the effect of LPS and IFN{gamma} on slfn gene expression in BMM from B6, IFN{alpha}/ßR–/– and IRF-1–/– mice, the cells were deprived of M-CSF (incubation in normal medium without L-cell supernatant) for 14 h, which induces growth arrest in G0 of the cell cycle (21,22). Subsequently, one set of resting cells was stimulated with LPS or IFN{gamma} for 12 h in growth medium still lacking M-CSF and thereafter processed for RNA isolation. Another set of M-CSF starved, G0 arrested BMM was restimulated with M-CSF to re-enter the cell cycle by change to L-cell supernatant containing growth medium. These cells were concomitantly stimulated with either LPS, IFN{gamma} or nothing and incubated for 12–14 h, the time point where DNA synthesis reached a maximum after restimulation with M-CSF (data not shown). The cells were subsequently processed for RNA isolation. To determine the time point of S-phase entry, cell proliferation of BMM was measured as described elsewhere (23). Shortly, 105 M-CSF starved BMM in 24-well plates were restimulated with L-cell supernatant containing growth medium in the presence or absence of either LPS or IFN{gamma}. Samples were obtained every 2 h by incubation of the cells in 0.5 ml of medium containing 1 µCi [3H]thymidine for 2 h and subsequent fixation in ice-cold 70% methanol. The fixed cells were washed with ice-cold 10% trichloroacetic acid (TCA) and solubilized in 1% SDS and 0.3% NaOH. The incorporated radioactivity was determined by liquid scintillation. Each time point was performed in triplicates. Maximal [3H]thymidine incorporation was noted ~14 h after restimulation with M-CSF. The M1 myeloblastic leukemia cell line (24) is competent for induction of terminal myeloid differentiation upon stimulation with IL-6 or leukemia inhibitory factor (LIF) (2528). The cells were induced for differentiation with IL-6 at 50 ng/ml (Sigma) or LIF at 25 ng/ml (Sigma) for 3 days. Peripheral T cells were purified from lymph nodes of B6 mice by nylon wool passage followed by depletion of residual MHC class II+ cells with magnetic beads (Miltenyi). These purified T cells were activated in culture wells (3–6 x 106 cells/well) of 6-well plates (Nunc) that had been coated with 10 µg/ml anti-CD3{varepsilon} mAb 145-2C11 (29) and 10 µg/ml anti-CD28 mAb 37.51 (30) for 1 or 2 days. Thymocyte subsets from B6 mice were prepurified by low tox rabbit plus guinea pig complement lysis (Cedarlane/Biozol, Eching, Germany) employing anti-CD4 mAb RL-174.2 (31), anti-CD8 mAb AD4-15 (32), or both mAbs. Subsequently, individual thymocyte subsets CD4+CD8CD3+ (CD4), CD8+CD4CD3+ (CD8), CD4+CD8+ (DP) and CD4CD8CD3 (DN) were isolated upon staining with anti-CD4 mAb GK1.5 (BD Biosciences, Heidelberg, Germany), anti-CD8a mAb 53-6.7 (BD Biosciences) and anti-CD3 mAb 145-2C11 (BD Biosciences) by sorting using a Becton-Dickinson FACStar.

Northern blot analysis and RT–PCR
Total splenic RNA was isolated from L. monocytogenes EGD infected mice using TRIZOL (Invitrogen, Karlsruhe, Germany) as recommended by the manufacturer. For northern blot analysis, 10 µg of total RNA were separated on 1.2% agarose/formaldehyde gels and transferred onto Hybond-N+ membrane (Amersham) by standard procedures. The blots were analyzed with randomly primed [{alpha}-32P]dCTP-labeled slfn gene specific, partial cDNA or murine ß-actin cDNA probes. Hybridization was performed overnight at 62–65°C in a solution containing 0.5 M NaPO4 pH 7.2, 7% SDS, 1 mM EDTA, 1% BSA, 100 µg/ml salmon sperm DNA. The blots were washed at 62–65°C with 2x SSC, 0.5% SDS (twice for 30 min) and subsequently with 0.2x SSC, 0.5% SDS for 30 min. Autoradiography was done using Hyperfilm-MP films (Amersham). Stripping of probed membranes was done as recommended by the manufacturer and equal loading of RNA was verified by reprobing the blots with ß-actin cDNA. For RT–PCR analysis of slfn gene expression, total RNA from thymocytes, peripheral T cells, M1 cells and BMM was isolated using the Trizol reagent. The purified RNA was digested with DNaseI and reverse transcribed with SuperScript II RT (Invitrogen, Karlsruhe, Germany). To normalize the cDNA content for further comparative analyses, a ß-actin specific PCR was performed with serial sample dilutions. A normalized amount of cDNA yielding equivalent amounts of ß-actin-specific PCR products were applied for semiquantitative PCR analysis. The PCR products were subjected to agarose gel electrophoresis and transferred to Hybond-N+ membrane (Amersham). For reduction of cycling numbers, to minimize overt non-linear amplification of the individual samples, the slfn-specific PCR products were detected with 32P-labeled oligonucleotide probes. The cycling numbers needed for detection were in the range of 21–24 cycles.

Primers used were: ß-actin forward 5'-TGGAATCCTGTGGCATCCATGAAC, ß-actin reverse 5'-TAAAACGCAGCTCAGTAACAGTCCG, slfn1 forward 5'-CCAGATGTCTCTGTTGGGAA, slfn1 reverse 5'-GCTAAGACATGAGGAGCTTG, slfn2 forward 5'-CAAGCCATCTTTGGGCTGCC, slfn2 reverse 5'-CTCTGGAAGAGCAGTCAGTG, slfn3 forward 5'-CGAACTTGTACAGAAGAATCAA, slfn3 reverse 5'-GTAAACCTCTTCACACAGCCA, slfn4 forward 5'-GCAGTTCCTCAAATCCAGAC, slfn4 reverse 5'-GTAAACCTCTTCACACAGCTG, slfn81 (B6) forward 5'-AGGCATGTATCAAATACAGGCCT, slfn8 (B6) reverse 5'-ACTGAGCCCCCATTGGTCTCAA, slfn8 (129) forward 5'-AGAAGGCATTTATCGAATACAGT, slfn8 (129) reverse 5'-ACTGAGCCCCCACTGGTCTTGT, slfn9 (B6) forward 5'-GGCATATATCAAATGCAGTCCG, slfn9 (B6) reverse 5'-ACTGAGCCCCCACTGGTCTTGT, slfn9 (129) forward 5'-AGAAGGCATATATCAAATGCAGT, slfn9 (129) reverse 5'-ACTGAGCTCCCACTGGTCTCGA, slfn10 (B6) forward 5'-GGGCAAGAAGAGATGTGTTAAG, slfn10 (B6) reverse 5'-GAGCCCCCATTAGTCTCAATAC, slfn10 (129) forward 5'-AAGGGCAAGAAGAGATGTGTTAAG, slfn10 (129) reverse 5'-GAGCCCCCATTAGTCTCAATAC, slfn5 forward 5'-GATTCTTGGTGACTCTGACTCGC, slfn5 reverse 5'-CTCTTTGGTGAGAACCCAGTGG. The olignucleotides used for 32P-labeled detection of PCR products were: slfn5 5'-GTGACGCTCTTCTGATTTCCCCGAAC, slfn8-10 5'-CCTCTACACCATCCTTGGGGAGCAGG, slfn1-4 5'-RTTCTGCTGYGCAGTRTTCKC.

Retroviral expression
The full-length coding slfn cDNAs were modified to code for an N-terminal fused myc-epitope (amino acid sequence MASDASMQKLISEENLG) directly in front of the authentic start-ATG codon. These fusion constructs as well as unmodified full-length coding cDNAs were cloned into the retroviral vector pMSCV2.2_IRES-GFP, kindly provided by Dr B. Sha (University of California, Berkeley, CA). The production of recombinant retrovirus in Phoenix-eco cells (33) was performed according to standard procedures. In brief, 4 x 106 Phoenix-eco cells were seeded in a 10 cm dish 1 day prior to transfection. Twenty micrograms of retroviral construct supplemented with 10 µg of pM13, driving the expression of gag-pol, were CaPO4 transfected. Retrovirus containing supernatants were harvested at 48 and 72 h after transfection, 0.4 µm filtered and stored at –80°C. For analysis of the impact of slfn gene expression on the growth of NIH 3T3 fibroblasts, subconfluent cells, at a density allowing unrestricted growth for another 3 days, were transduced for 6 h. The GFP reporter expression, driven by the internal IRES sequence, as well as the expression of the myc-epitope tagged slfn proteins, was analyzed 36 h after transduction. Whole cell extracts prepared in Laemmli sample buffer were separated by SDS–PAGE, western blotted and myc-tagged proteins were revealed using the anti-human myc mAb 1-9E10.2 (ATCC CRL-1729), peroxidase conjugated goat anti-mouse IgG (Dianova, Hamburg, Germany) and ECL-western blot detection reagents (Amersham, Buckinghamshire, UK). The number of adherent, living cells was determined 3 days after transduction by trypan blue exclusion and the percentage of living cells compared to pMSCV2.2_IRES-GFP control transduced cells was calculated. Individual experiments were set up in triplicates and repeated twice.

Generation and analysis of slfn8 transgenic mice
A slfn8 cDNA was inserted into the VA CD2 cassette (34), which drives transgene expression predominantly in the T cell compartment and in B cells at early stages of development (35). After removal of prokaryotic vector sequences, the VA CD2-slfn8 construct was injected into CBAxC57BL/6 F2 embryos, yielding three transgene expressing founders. The expression levels of the transgene were comparable in those individual lines, being 2–4-fold elevated in the thymus and 2-fold elevated in peripheral resting T cells over endogenous levels as determined by quantitative PCR analysis (data not shown). The analysis of the individual transgenic lines revealed a corresponding phenotype with comparable expression. Therefore, a representative transgenic line was used for further experiments. This slfn8 transgenic line was maintained by backcrossing to B6 mice and after backcrossing at least five times, 6–8-week-old mice were used for analysis. For FACS analysis, thymocytes, splenocytes or lymph node cells were stained with anti-CD4–FITC or –Cy5 (mAb YTS 191), anti-CD8{alpha}–FITC, –PE or –Cy5 (mAb YTS 169), anti-CD3{varepsilon}–FITC (Pharmingen), anti-CD44–Cy5 (mAb IM7), anti-CD25–PE (mAb PC61) and anti-CD62L–FITC (mAb Mel-14). For proliferation assays, lymph node T cells from transgenic mice or littermates were purified with anti-CD4 and anti-CD8 magnetic beads (Milteny Biotec) yielding a purity of >96%. Triplicates of these T cells (4 x 105 cells/well) were stimulated for 72 h with titrated amounts of plate-bound anti-CD3{varepsilon} mAb 145-2C11 (29) and 10 µg/ml soluble anti-CD28 mAb 37.51 (30) in 96-well flat bottom plates. PMA (Sigma) and ionomycin (Sigma) were used for stimulation of T cells at 50 ng/ml and 1 µM, respectively. [3H]Thymidine incorporation of T cells was determined by addition of 1 µCi/well [3H]thymidine (Amersham) for 8 h and measured using a TopCount NXTTM ß counter (Packard Instrument Co.). Alternatively, purified peripheral T cells were incubated with 5 µM CFSE (Molecular Probes) in PBS for 10 min at 37°C, stimulated with plate-bound anti-CD3{varepsilon} mAb 145-2C11 and soluble anti-CD28 mAb 37.51 as described above and incubated for 72 h as indicated.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Identification of new slfn family members constituting a distinct subgroup
To identify genes involved in the immune response against intracellular bacteria, we generated by suppression-subtractive hybridization a differential cDNA library derived from T and B cell depleted splenocytes from L. monocytogenes infected B6 mice 2 days p.i. with reference to cells from uninfected animals (19). Within this library, we identified partial slfn4 cDNAs and one with a putative open reading frame encoding part of a protein with significant homology to the C-terminal regions of slfn3 and slfn4, but extending for a further ~400 amino acids. After performing 5'-RACE we cloned a cDNA encoding a putative protein of 910 amino acids in length which was designated slfn8. Pairwise BLAST alignment showed that the first 570 amino acids were significantly homologous to slfn3 and slfn4 (30% identity, 50% similarity) while the remaining region was unique to slfn8.

For further characterization of the genomic organization of the slfn8 gene, a murine genomic 129/Ola cosmid library was screened by PCR. While two isolated cosmids gave positive PCR products with every slfn8 cDNA sequence based primer combination applied, two additional cosmids produced variable results. Sequencing of these four cosmids revealed overlapping coverage of ~125 kbp, as indicated in Fig. 1(A). Within the first 10 kb, we identified the slfn2 gene. Furthermore, our analysis revealed the slfn8 gene and three additional slfn8 homologous genes which were designated slfn5, slfn9 and slfn10 (Fig. 1A and B). No potential intervening genes were identified. Subsequently, the expression of these newly identified slfn genes was verified by RT–PCR using cDNA generated from IFN{gamma}-stimulated BMM from B6 and 129 mice (see also below) and full-length products were cloned. Taken together, our analyses revealed that the slfn5, slfn8, slfn9 and slfn10 genes each harbour four analogously organized, coding exons, whereby the last two correspond to the part of the proteins which is unique to this third subgroup within the Slfn protein family (Fig. 1A and B). Slfn8, slfn9 and slfn10 show a remarkable degree of homology with each other, ranging in 85–87% identity over the complete protein sequence. In contrast, slfn5 is rather distantly related to the former group, in light of 40% identity and 60% similarity compared to the others. However, conserved short blocks within all known slfn proteins as well as within the region that characterizes the subgroup III members are obvious (Fig. 2). The search for functionally relevant motifs, using the Prosite, BLOCKS and Pfam databases (www.mgd.ahc.umn.edu/panal/), revealed the presence of an ATP/GTP binding domain (P-loop) (Fig. 2; motif I) and a DEAQ box containing Walker B motif (Fig. 2; motif II) in the C-terminal third of the molecules, indicative of ATPase activity (3638). Running the sequences against the NCBI conserved domain database (CDD) revealed weak, albeit significant, homology to motifs typical for RNA/DNA helicases. Helicases belonging to the superfamily I commonly contain a set of eight consecutive characteristic motifs (3942). Analysis of the amino acid sequences distal to the ATP binding motifs revealed the presence of conserved blocks that show striking homology, also regarding the spacing of the individual motifs, to the signature of superfamily I helicases (Pfam01443), especially to RNA helicases from single stranded RNA viruses (Fig. 2). Furthermore, common to all Slfn proteins is a conserved region in the N-terminal third of the molecules being significantly homologous to part of a conserved domain signature (CDD: COG2865) present in a variety of prokaryotic and eukaryotic putative proteins which are predicted to be transcriptional regulators or helicases (Fig. 2). Taken together, the analysis of the deduced amino acid sequences of the new Slfn protein family members suggests that these proteins have RNA helicase or RNA structure modelling activity, essential functions in RNA metabolism.



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Fig. 1. Genomic configuration of the slfn5, slfn8, slfn9 and slfn10 gene cluster. (A) The positions of the exons of the slfn2, slfn5, slfn8, slfn9 and slfn10 genes are depicted based on the sequence analyses of the indicated overlapping cosmid inserts identified from a 129/Ola-derived genomic cosmid library. Arrows indicate the transcriptional sense orientation of the individual genes. The slfn5, slfn8, slfn9 and slfn10 genes each harbour four coding exons. (B) A schematic depiction of the three subgroups within the slfn protein family aligned according to overall homology of exon encoded regions. Subgroup I comprises proteins slfn1 and slfn2, subgroup II comprises slfn3 and slfn4, and the putative proteins slfn6 and slfn7 (14). Subgroup III comprises the newly identified slfn5, slfn8, slfn9 and slfn10 proteins. The triangles indicate exon boundaries.

 


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Fig. 2. Alignment of amino acid sequences of Slfn1, Slfn4 and members of the subgroup III of the Slfn protein family. The sequences were aligned using the CLUSTAL_W 1.5 multiple sequence alignment program. Conserved amino acids are shaded in black and grey, indicating identity or similarity, respectively. Exon boundaries are indicated by triangles. The dashed underlined, conserved region, present in all Slfn proteins, indicates homology to part of a conserved domain signature (NCBI, CDD: COG2865) found in a variety of putative proteins assigned as transcriptional regulators or helicases. The helicase superfamily I signature motifs (39,41,42) are depicted below the underlined and numbered, helicase-like activity classifying regions in Slfn5, Slfn8, Slfn9 and Slfn10. These signatures indicate conserved amino acid residues; +, a hydrophobic residue; O, hydrophilic residue; X, any residue. Precedents for deviations in the conserved positions are not uncommon within helicase-classified proteins (39) and slfn protein analogous variations can be found within the group of viral RNA helicases (Pfam01443). In addition, the short, dotted underlined region of the Slfn subgroup III proteins exhibits homology to a correspondingly positioned region in UvrD DNA helicases (Pfam00580). The region underlined and designated Q marks the putative position of the recently identified Q-motif of DEAD box helicases (63). The positions of the conserved glutamine and an upstream characteristic phenylalanine are indicated (*).

 
Induction of slfn gene expression upon infection
Conceptually, the identification of slfn8 was based on its differential expression in the spleens of L. monocytogenes infected versus non-infected mice. Therefore, splenic mRNA expression of slfn genes was analyzed in RAG1–/–, B6, TNFRI–/– and IRF-1–/– mice 2 days after infection with L. monocytogenes. Significant upregulation of mRNA expression was obvious for slfn1, slfn2, slfn4, slfn5, slfn8, slfn9 and slfn10, in B6 and RAG–/– mice (Fig. 3). The upregulation of expression was not compromised in Listeria infected TNFRI–/– mice, suggesting that induced TNF{alpha} does not markedly contribute to the induction of slfn expression via the TNF{alpha} p55 receptor. However, markedly reduced levels of slfn mRNA levels with the exception of slfn2 were evident in IRF-1–/– mice, indicating that the transcription factor IRF-1 participates in the transcriptional control of most analysed slfn genes at least after listerial infection. IFN{gamma}R–/– mice also failed to upregulated slfn5, slfn8, slfn9 and slfn10 expression upon listerial infection (data not shown), suggesting that IFN{gamma} participates in the control of slfn gene expression.



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Fig. 3. Expression of slfn genes in splenocytes of L. monocytogenes infected mice. Groups of RAG–/–, TNFRI–/–, IRF-1–/– and B6 mice were infected with L. monocytogenes (5 x 103 i.v.). Total splenic RNA was isolated 2 days post infection and subjected to northern blot analysis. The blots were analyzed with slfn gene or ß-actin specific cDNA probes. Normalization of the samples was controlled by rehybridization of the blots with a ß-actin specific-probe.

 
Expression of slfn genes in macrophages
The upregulation of slfn gene expression in infected RAG–/– mice suggested an involvement of non-lymphoid splenocytes, e.g. macrophages. Establishment of the effector functions of macrophages upon activation is associated with growth arrest. Previous work suggested participation of slfn family members in the regulation of the cell cycle (14). Therefore, we analyzed the expression of slfn genes in proliferating and growth arrested BMM. A G0 cell cycle arrest can be induced in BMM by M-CSF withdrawal (Fig. 4A) (21,22). After restimulation with M-CSF these resting cells re-enter the cell cycle synchronously and maximal DNA synthesis is observed after 10–12 h (Fig. 4A) (21,22). Furthermore, stimulation of BMM with LPS or IFN{gamma} not only activates effector functions but also induces a growth arrest in the early G1 stage or in the G1/S transit of the cell cycle (23). We examined slfn gene expression in BMM, stimulated by LPS, IFN{gamma} or not, which had been arrested by M-CSF withdrawal or were subsequently M-CSF restimulated to re-enter the cell cycle. Remarkably, stimulation with LPS or IFN{gamma} led to significant upregulation of the expression of most slfn genes in B6 macrophages independent from the cell cycle state (Fig. 4B). Only slfn3 expression was refractory to these stimuli and expression of slfn4 was only elevated by LPS, but not by IFN{gamma} stimulation. Importantly, we noted no considerable differences in slfn gene expression comparing growth arrested with S-phase macrophages (Fig. 4B). Identical results were obtained with post S-phase (20 h after M-CSF restimulation) and asynchronously growing macrophages (data not shown). Taken together, our results indicate that slfn gene expression in BMM is not regulated throughout the cell cycle. Parallel analysis of IFN{alpha}/ßR deficient BMM revealed LPS mediated responses not obscured by events induced by autocrine effective type I IFNs (43,44). These cells did not upregulate slfn gene expression in response to LPS, although growth was arrested as in wild-type macrophages. Obviously, autocrine type I IFNs induced elevated levels of slfn gene expression upon stimulation of BMM with LPS. IRF-1 seemed to participate in the transcriptional control of slfn gene expression (Fig. 3). Hence we examined BMM derived from IRF-1–/– mice. Increased expression of slfn genes was not observed in IRF-1–/– BMM upon IFN{gamma} stimulation, revealing transcriptional IRF-1 dependency (Fig. 4B). In analogy to LPS stimulated IFNR{alpha}–/– BMM, the IFN{gamma} induced growth arrest in IRF-1–/– BMM was not paralleled by upregulation of slfn gene expression. We concluded that increased expression of any slfn gene is not essential for growth arrest in macrophages induced either by M-CSF withdrawal or by LPS or IFN{gamma} stimulation. Therefore, our results argue against an obligatory role of Slfn proteins in cell cycle control in LPS or IFN{gamma} activated macrophages.



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Fig. 4. Expression of slfn genes in macrophages. (A) DNA synthesis in BMM derived from B6, IFNR{alpha}–/–, or IRF-1–/– mice was assessed by incorporation of [3H]thymidine after induction of growth arrest by M-CSF withdrawal (– M-CSF), and during subsequent restimulation with M-CSF (+ M-CSF). Incorporation of [3H]thymidine was measured every 2 h upon M-CSF restimulation to determine the time needed to enter the S-phase of the cell cycle. Shown are the values of maximal [3H]thymidine incorporation which were reached ~12 h after M-CSF restimulation. Concomitant activation of macrophages with LPS (+ M-CSF/LPS) or IFN{gamma} (+ M-CSF/IFN{gamma}) induced growth arrest reflected by significantly decreased incorporation of [3H]thymidine. (B) The relative expression levels of slfn genes in growth arrested or proliferating BMM from B6, IFNR{alpha}–/– or IRF-1–/– mice were analyzed by semi-quantitative PCR. Macrophages were growth arrested by M-CSF withdrawal overnight and subsequently stimulated with LPS, IFN{gamma} or left untreated for 12 h in medium lacking M-CSF (G0). A second set of growth arrested macrophages was restimulated with M-CSF to re-enter the cell cycle. Concomitantly, macrophages were stimulated with LPS, IFN{gamma} or not for 12 h, the time point of maximal DNA synthesis. The PCR products were separated by agarose gel electrophoresis, blotted and detected by hybridization with 32P-labeled oligonucleotide probes. (C) Differential expression of slfn genes in M1 cells undergoing terminal myeloid differentiation. M1 cells were stimulated with IL6, LIF or not for 3 days and slfn gene expression was analyzed as described above.

 
In addition to a role of Slfn proteins in LPS or IFN{gamma} induced macrophage activation, an increased expression level of slfn genes in the spleens of infected RAG–/– mice could also be associated with differentiation of recruited monocytes/macrophages. The monocytic leukemia M1 cell line is widely used for analysis of processes governing terminal myeloid differentiation (45). When treated with IL6 or LIF, M1 cells differentiate into growth arrested, macrophage-like cells (2528). Analysis of slfn gene expression in undifferentiated versus IL6 or LIF treated M1 cells revealed that macrophage-like differentiation is accompanied by marked transcriptional upregulation of almost all slfn genes except slfn3 and slfn9 (Fig. 4C). Hence, slfn proteins may not only take part in lymphoid (14), but also in myeloid, development and differentiation.

Ectopic slfn expression in fibroblasts
To assess more directly whether Slfn proteins influence cell growth, we expressed Slfn1, Slfn5, Slfn8, Slfn9 and Slfn10 by retroviral transduction in NIH 3T3 fibroblasts. The individual myc-tagged slfn cDNAs were introduced into the retroviral vector pMSCV2.2-IRES-GFP, which expresses a bicistronic mRNA encoding the marker green fluorescent protein (GFP). Subconfluent NIH 3T3 cells were efficiently infected with high titer retroviral stocks (Fig. 5) and numbers of viable cells were assessed 3 days later. Consistent with the previously noted anti-proliferative activity (14) we observed that expression of full-length Slfn1 led to a significantly reduced recovery in viable cell numbers compared to control vector transduced cells. Slfn1 expressing cells stopped dividing and eventually died with signs of apoptosis (data not shown). This resulted 3 days after transduction in viable cell numbers that were actually lower than originally seeded. In contrast, the expression of a Slfn1 mutant, lacking the first 27 amino acids, but expressed at an even higher level than full-length Slfn1, was tolerated and growth of fibroblasts was unaffected. This indicates that the anti-proliferative effect observed for full-length Slfn1 seems to depend on integrity of its N-terminal region and reflects specificity of the processes initiated by slfn1 expression. In contrast, no anti-proliferative activity was evident for Slfn5, Slfn8, Slfn9 or Slfn10 (Fig. 5). Furthermore, we could establish stable cell lines by FACS sorting of GFPhigh cells expressing correspondingly high levels of Slfn5, Slfn8, Slfn9 or Slfn10. These cell lines showed growth characteristics indistinguishable from normal NIH 3T3 cells (data not shown). However, establishment of cell lines expressing even minute amounts of full-length Slfn1 constantly failed. Collectively, we noted no direct influence of Slfn5, Slfn8, Slfn9 or Slfn10 on growth of fibroblasts.



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Fig. 5. Ectopic expression in fibroblasts and analysis of growth. Subconfluent NIH 3T3 fibroblasts were retrovirally transduced with vectors encoding full-length slfn1, an N-terminal deletion variant of slfn1, slfn5, slfn8, slfn9 or slfn10. Transduction efficiency and expression was monitored by FACS analysis 36 h after infection by detection of marker GFP expression, which is encoded by the bicistronic retroviral mRNA. Expression of myc-tagged slfn proteins was analyzed 36 h after transduction by western blot analysis of whole cell lysates upon loading of equal cell number equivalents and usage of the anti-myc mAb 1-9E10. Three days after infection viable cell numbers were determined by trypan blue exclusion. The experiments were set up in triplicate. Identical results were obtained in three independent experiments and also by expression of slfn proteins without a myc-tag.

 
Expression of slfn genes in T cells
The members of the subgroups I and II of the slfn gene family (Fig. 1B) are differentially expressed during the development of thymocytes and upon activation of peripheral T cells (14). We next examined the expression of the third slfn subgroup members, namely slfn5, slfn8, slfn9 and slfn10, in thymocyte subsets and in purified lymph node-derived T cells after CD3/TCR stimulation. Examination of slfn1, slfn2, slfn3 and slfn4 expression was included in our analysis. As was shown previously, slfn1 and slfn4 are differentially expressed during thymocyte development (Fig. 6A) (14). In contrast, we found relatively invariant levels of slfn5, slfn8, slfn9 and slfn10 expression in immature CD3CD4CD8 (DN), CD4+CD8+ double-positive stage (DP) and mature CD4+ or CD8+ thymocytes (Fig. 6A). However, the slight reduction of slfn8 and slfn9 mRNA levels at the DP stage was consistently observed in independent experiments. The same was true for the slight increase in slfn10 and slfn5 expression in the DN or DP stage, respectively. In contrast to thymocytes representative for distinct developmental stages, a differential regulation of expression was evident in purified peripheral T cells upon stimulation by TCR ligation (Fig. 6B). We observed significant downregulation of slfn5 and slfn8 expression, while slfn9 expression increased concomitantly. Interestingly, the expression level of slfn10 remained rather constant. Since IFN{gamma} is an inducer of slfn gene expression in macrophages, we analyzed in addition whether autocrine IFN{gamma}, produced upon activation of T cells, influenced the slfn gene expression profile. Abrogation of IFN{gamma} responsiveness did not lead to an altered expression pattern of slfn genes in IFN{gamma}R deficient T cells (Fig. 6B), suggesting that IFN{gamma} induced signalling did not overtly influence transcriptional control of slfn gene expression in activated T cells.



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Fig. 6. Differential expression of slfn genes in thymocytes and peripheral T cells. The relative expression levels of slfn genes were assessed in subsets of thymocytes and purified peripheral T cells by semi-quantitative RT–PCR analyses using normalized cDNA samples. Purified thymocytes subsets were immature CD3CD4CD8 (DN), CD4+CD8+ double-positive stage (DP), and mature single positive CD4+ (CD4) or CD8+ (CD8) thymocytes. Purified lymph node T cells from either B6 or IFN{gamma}R–/– mice were activated by TCR/CD28 stimulation for 1 and 2 days or not. Detection of transcripts specific for the type I and type II IFN inducible 47 kDa GTPase IIGP reflects autocrine IFN stimulation in activated peripheral T cells (19). The PCR products were separated by agarose gel electrophoresis, blotted and detected by hybridization with 32P-labeled oligonucleotide probes. Analyses were repeated giving identical results.

 
Slfn8 transgenic mice
To gain deeper insights into the physiological significance, especially of Slfn subgroup III family members, we generated transgenic mice carrying a slfn8 cDNA under the control of the T-cell specific hCD2 locus control region (34). Analysis of the T cell compartment revealed a significant reduction in thymic cellularity of ~60% in slfn8 transgenic animals compared to littermates (Fig. 7B). However, the CD8 and CD4 surface marker profile of slfn8 transgenic thymocytes did not reveal gross differences compared to littermate controls, suggesting that transgenic slfn8 expression did not overtly compromise thymocyte maturation. Immature thymocytes can be subdivided by surface expression of CD44 and CD25. The most immature are CD44+CD25 (DN1), which subsequently develop into CD44+CD25+ (DN2), CD44CD25+ (DN3) and CD44CD25 (DN4) subsets prior to expression of CD8 and CD4 (3). The hCD2 cassette driven transgene expression in thymocytes starts between the DN1 and DN3 stage (35,46). In terms of absolute cell numbers the generation of DN3 thymocytes was only slightly affected by transgenic slfn8 expression, whereas DN4 thymocytes were significantly decreased in slfn8 transgenic mice compared to littermate controls (Fig. 7B). Apparently, ectopic expression of slfn8 partially blocked DN3 to DN4 transition of developing thymocytes, providing an explanation for the reduced thymocyte numbers in slfn8 transgenic mice. In contrast, we did not note obvious changes in cellularity or surface marker profiles (for CD4, CD8, Fig. 7A; for CD62L, CD44, CD69, data not shown) of lymph node or splenic T cells from slfn8 transgenic mice. To assess the response to TCR stimulation in vitro, purified peripheral T cell from slfn8 transgenic and littermate animals were stimulated with plate-bound anti-CD3{varepsilon} mAb and proliferation was measured by [3H]thymidine incorporation. Compared to littermate controls the slfn8 transgenic T cells exhibited a significantly reduced proliferative response, which was not compensated by costimulation of CD28 and was also obvious upon stimulation with PMA and ionomycin (Fig. 7C). Labeling purified T cells with CSFE prior to in vitro stimulation revealed that the fraction of cells starting to divide as well as the number of cell divisions performed is significantly reduced in slfn8 transgenic T cells compared to littermate controls (Fig. 7C). Interestingly, this effect was observed for both CD4+ and CD8+ T cells, indicating that transgenic slfn8 expression does not affect these subsets specifically. Taken together, transgenic expression of slfn8 in the T cell compartment negatively modulated pre-T cell development and led to a reduced proliferative response of peripheral T cells upon TCR stimulation.



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Fig. 7. T cell development Slfn8 transgenic mice Transgenic expression of slfn8 impairs thymocyte development. (A) Developmental block at the CD44CD25+ DN3 stage of immature, slfn8 transgenic thymocytes. Flow cytometric analysis of thymocytes and peripheral T from mice with the indicated genotype. Representative dot plots of total thymocytes, lymph node cells or splenocytes stained for CD4 and CD8. Immature thymocytes were analysed by gating on CD3CD4CD8 cells and staining for CD44 and CD25. (B) Absolute numbers of thymocyte subpopulations with the indicated genotypes. Shown are the mean ± SD with n = 16 for total thymocytes and n = 6 for immature DN thymocyte analyses. (C) Reduced proliferation of slfn8 transgenic peripheral T cell upon TCR mediated stimulation. Purified T cells from slfn8 transgenic mice and littermate controls were cultured for 72 h with the indicated doses of plate-bound anti-CD3 mAb with or without addition of soluble anti-CD28 mAb or stimulated with PMA/ionomycin. The cells were pulsed with [3H]thymidine and incorporation was determined. (D) Proliferation profiles of CFSE-labeled, purified T cells stimulated in vitro with 4 µg/ml plate-bound anti-CD3 mAb with addition of 10 µg/ml soluble anti-CD28 mAb for 24 h, 48 h and 72 h. The cells were stained with anti-CD8–Cy5 or anti-CD4–Cy5 plate-bound anti-CD3 mAb with or without addition of soluble anti-CD28, and percentages of CFSE-labeled T cells that have undergone one or more divisions are shown.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Members of the Slfn protein family have been implicated in regulatory processes governing lymphocyte differentiation and growth control (14). We have identified four new members of the slfn gene family which constitute a distinct, third subgroup. In contrast to the previously described Slfn proteins, the newly identified family members harbour sequence motifs indicative for ATP binding in the context of an RNA helicase-like activity. Most slfn genes are strongly induced in the spleen of mice infected with the intracellular bacterial pathogen L. monocytogenes. They are differentially regulated in developing thymocytes, in activated peripheral T cells, in LPS or IFN{gamma} activated macrophages or during terminal myeloid differentiation of M1 leukemia cells. Transgenic expression in mice revealed that dysregulated expression of slfn8 negatively influences thymocyte development and impairs the response of peripheral T cells to TCR mediated stimulation. Thus, these new Slfn subfamily members are integrated into regulatory networks that control T cell development and activation.

The actual molecular function of Slfn1, the previously identified prototype of this protein family, as well as of the subgroup II family members is unknown (14). The sequence analysis of the newly identified family members Slfn5, Slfn8, Slfn9 and Slfn10 provides a first functional indication. The C-terminal thirds of these molecules, which are unique to the third subgroup within the Slfn protein family, harbour typical Walker A and B motifs, the latter with a DEAQ box, indicative of ATP binding (Fig. 2; motifs I, Ia, Ib and II) (3638). The degree of homology and spacing of consecutive, conserved motifs present in Slfn5, Slfn8, Slfn9 and Slfn10, suggests that the third Slfn family subgroup represents a new subfamily of RNA-dependent ATPases belonging to the helicase superfamily I (39,40,42). Although structural conservation of RNA helicases is reflected by the presence of conserved amino acids within the RNA helicase fingerprint motifs (Fig. 2; see consensus motifs III–VI) (41,42), some variations in the motifs present in the newly identified Slfn proteins are noticeable. However, the presence of leucine instead of the conserved glycine in motif III or of serine instead of the conserved glutamine or lysine in motif V are not without precedent (see viral RNA helicases, PFAM 01443). In general, helicase motifs III–VI seem to be structurally conserved elements that are rather unique to individual helicase families or individual members probably dictated by specific functions, like DNA/RNA target recognition or interaction with accessory factors (41). RNA helicases perform essential functions in RNA metabolism, as modulators of RNA secondary structures being involved in gene specific transcription, pre-mRNA processing, RNA export, translational initiation, or RNA degradation (42,47). For example, mda-5, an interferon-inducible DExH RNA helicase, is implicated in cellular differentiation and apoptosis (48), and the helicase MCM5 participates in Stat1 mediated transcriptional control (49). The restricted expression of slfn genes, preferentially in hematopoietic cells (14) (data not shown), suggests that Slfn proteins participate in the regulation of cell type and differentiation-stage specific gene expression. Our analysis of the genomic organization of the slfn5, slfn8, slfn9 and slfn10 genes revealed localization within a single cluster proximal to the slfn2 gene previously mapped to chromosome 11 (48.0 cM) (14,50,51). These slfn genes are localized within a 1.4 Mb region, exhibiting conservation of synteny to human chromosome 17q11-17q21.1 and confined by the markers Scya2 and D11Pas18 (50,51), which harbours the as yet unidentified Ovum mutant locus. This single genetic trait is responsible for the DDK syndrome describing embryonic lethality of F1 embryos from crosses between murine DDK females and non-DDK males (52). However, involvement of any slfn gene in this developmental disorder remains to be determined.

The stimuli governing transcriptional control of slfn gene expression seem to be complex. During thymocyte development, marked differential expression is obvious for slfn1, slfn2 and slfn4 (14), while the expression levels of the newly identified slfn gene members, slfn5, slfn8, slfn9 and slfn10, fluctuate only moderately. In contrast, upon TCR mediated activation of peripheral T cells, differential downregulation of slfn5 and slfn8, and upregulation of slfn9 expression was noted, while the expression of slfn10 remained rather unchanged. While this proceeds independently of IFN{gamma} produced upon T cell activation, the stimulation with IFN{gamma} markedly increased expression of almost all slfn genes, except slfn3 and slfn4, in resting and proliferating BMM. Under these conditions, transcriptional upregulation in BMM depends on the transcription factor IRF-1. However, the type-I IFN mediated upregulation of slfn gene expression upon stimulation of resting or proliferating BMM with LPS proceeds independently of IRF-1. Consistent with the role of IRF-1 for IFN{gamma} induced transcriptional regulation, we found that the expression of slfn1, slfn4, slfn5, slfn8, slfn9 and slfn10 is significantly impaired in splenocytes of IRF-1–/– mice upon infection with L. monocytogenes. The comparable degree of increased slfn gene expression in infected RAG1–/– mice suggests that the transcriptional upregulation observed in B6 mice is attributable to a certain extent to expression in non-lymphoid cells, possibly NK cells, dendritic cells (various slfn genes are expressed in both cell types; data not shown) or macrophages. Furthermore, a possible role in myeloid differentiation was revealed by our finding of a concerted upregulation of slfn gene expression, except slfn3 and slfn9, in myeloblastic M1 cells undergoing IL6 or LIF induced terminal differentiation into macrophage like cells. This STAT3 dependent process is accompanied by upregulation of IRF-1 expression (45,53), which participates in the induction of the growth arrest (54) and could be involved in the differential expression of slfn genes during differentiation of M1 cells. Taken together, various stimuli govern differential expression of individual slfn genes during development, differentiation or activation of distinct hematopoietic cells, as shown for T cells and macrophages. We assume a critical role for Slfn proteins in the acquisition of cell-type specific properties and therefore significance for the processes in hematopoietic cells that are activated during terminal differentiation in the course of an immune response.

An involvement of Slfn proteins, especially of Slfn1 and implicitly of Slfn2 and Slfn3, in negative regulation of cell growth, was previously proposed, because ectopic expression of Slfn1 caused cell cycle arrest and high expression correlates with resting cellular states in T cells (14). Our analyses not only confirm the anti-proliferative activity exerted by Slfn1, but also show that the integrity of the N-terminal portion of the protein is essential to mediate this function. The mechanisms underlying the growth-inhibitory effects induced by IFNs or LPS in macrophages are still incompletely understood, although some regulatory elements, like cyclin D1, c-myc, mad1, p27kip1 or cdk4 have been identified (5559). The induction of an IFN{gamma} or LPS mediated G1 cell cycle arrest in M-CSF stimulated BMM was found to be uncoupled from upregulation of slfn gene expression in macrophages derived from either IRF-1–/– or IFN{alpha}/ßR–/– mice, respectively. This excludes an essential function for any Slfn protein, including Slfn1, as well as for IRF-1 or IFN{alpha}/ßR mediated signalling, in either IFN{gamma} or LPS induced growth arrest in macrophages. While these findings do not preclude functional interference with the cell cycle control machinery, an anti-proliferative activity of Slfn5, Slfn8, Slfn9 and Slfn10 was also not revealed upon ectopic expression in fibroblasts. However, based on the consequences of transgenic slfn8 expression in mice, we do not exclude a cell-type specific influence on growth control in T cells. These transgenic mice express slfn8, driven by an hCD2 cassette, in a preferentially T-cell specific fashion. Despite continuous expression of endogenous slfn8 throughout thymocyte maturation, ectopic expression starting in immature DN1-3 stage progenitors (35), interfered with thymic development. The cellularity in hemizygous transgenic mice was decreased, based on a developmental block at the DN3 stage resulting in reduced, presumably limiting numbers of DN4 stage immature thymocytes. Consequently, the generation of DP and SP thymocytes, although phenotypically inconspicuous, was quantitatively impaired. In addition, it cannot be excluded that the proliferative expansion, which accompanies DN4 to DP development (12), was likewise affected by transgenic slfn8 expression, based on our finding that the expression level of endogenous slfn8 was slightly reduced in DP compared to the more immature DN thymocytes. An increased rate of cell death in the thymi of slfn8 transgenic animals could not be detected by various means (data not shown), further suggesting that the reduced thymic cellularity is the consequence of a reduced rate of expansion instead of compromised viability. Interestingly, the proliferative response to TCR ligation of slfn8 transgenic peripheral T cells was significantly impaired. A substantially reduced fraction of cells enters the cell cycle and the number of divisions is also reduced upon stimulation of T cells expressing the slfn8 transgene. Endogenous slfn8 expression is normally downmodulated in activated T cells. Obviously, the continuous transgenic expression of slfn8 alters the proliferative potential of CD3-stimulated T cells. This could be due to a cell-type specific effect of slfn8 affecting cell cycle control in T cells or to an interference with the proliferation inducing signal cascade. Thus, subverting the finely tuned expression of slfn8 in developing and mature T cells profoundly alters physiologically relevant features. Future studies are directed to elucidate the molecular basis of the altered T cell physiology in slfn8 transgenic mice.

Although we still do not fully understand the exact function(s) of Slfn proteins, our findings emphasize previous notions of an regulatory role of members of this protein family in hematopoietic cells (14). Notably, the identification and initial functional analysis of a new subfamily comprising at least four members harbouring RNA helicase-like fingerprint motifs provides first clues on their activity and significance for the physiology of hematopoietic cells. The consequences of dysregulated expression of slfn genes are remarkable, exemplified by profoundly impaired pre-T cell development and functionality of mature T cells. The regulatory networks guiding both thymic development and differentiation of T cells upon antigenic stimulation are complex and exerted on multiple levels (1,3,6062). The obvious contribution of Slfn proteins in these processes necessitates the in-depth understanding of their functionality and will improve the molecular understanding of hematopoietic cell development and homeostasis.


    Acknowledgements
 
We are grateful to Toralf Kaiser, Dimitri Kioussis, Bill Sha and Carol Stocking for technical support and provision of vectors or cells. We thank Ute Guhlich, Jessica Bigott and Sandra Leitner for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft through Grant Ze363/2–1,2 (J.Z.).


    Abbreviations
 
BMM   bone marrow-derived macrophages
CDD   conserved domain database
GFP   green fluorescent protein
LIF   leukemia inhibitory factor
M-CSF   macrophage colony stimulating factor
TCA   trichloroacetic acid
TCR   T cell receptor

    Notes
 
Transmitting editor: T. Hünig

Received 5 April 2004, accepted 5 August 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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