Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22−/−[B6]) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag–specific B cells in IgTg hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance.
Molecules that regulate lymphocyte homeostasis, proliferation, and survival operate in concert to enable robust adaptive immune responses to foreign antigens (Ags). For the B cell lineage, the optimal outcome of these processes is a diverse antibody (Ab) repertoire purged of pathological (self-reactive) B cells. The elimination of pathological B cells occurs either through clonal deletion or receptor editing during B lymphopoiesis in the bone marrow, or in the periphery through the induction of anergy (Goodnow et al., 1988; Nemazee and Bürki, 1989; Gay et al., 1993; Tiegs et al., 1993). Anergic B cells primarily inhabit the spleen, are short-lived, and undergo activation-induced cell death (AICD) in response to B cell Ag receptor (BCR) stimulation (Goodnow et al., 1995; Shlomchik, 2008).
BCR ligation by agonistic anti-IgM Abs induces 30–50% of spleen B cells from WT mice to blast and undergo proliferation ex vivo (DeFranco et al., 1982). However, the threshold for B cell AICD can be influenced by genetically altering the stimulatory and inhibitory pathways that regulate BCR-induced activation (Inaoki et al., 1997). The B cell–restricted surface protein CD22 is generally considered to negatively regulate BCR signaling by recruiting potent intracellular phosphatases after BCR ligation (Doody et al., 1995; O’Keefe et al., 1996; Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., 1997; Tedder et al., 1997; Poe et al., 2000), and CD22−/− mice produce augmented levels of isotype-switched auto-Abs against DNA and some protein Ags (O’Keefe et al., 1999; Poe et al., 2011). Nevertheless, B cells from inbred CD22−/− mice with a B6/129 genetic background (CD22−/−[inbr]) are phenotypically and functionally normal ex vivo (Poe et al., 2004). In contrast, spleen B cells from C57BL/6 (B6) mice genetically deficient in CD22 (CD22−/−[B6]) undergo AICD after BCR stimulation (Poe et al., 2004), which is likely to be a result of their inability to induce c-Myc transcription factor expression that balances B cell proliferation versus AICD (Donjerković and Scott, 2000; Poe et al., 2004). These striking phenotypic differences in B cells between mouse lines with a common deletion of Cd22 indicate that important B cell signaling events that promote AICD are influenced differently by the B6 and 129 genetic backgrounds. These two CD22−/− mouse lines were therefore used to identify genetic and molecular factors regulating B cell AICD.
In these studies, a forward genetic screen was used to identify an evolutionarily conserved single-stranded RNA (ssRNA) binding protein, EndoU, as a novel regulator of AICD in CD22−/−[B6] mice. EndoU was also overexpressed by anergic peripheral B cells from double-transgenic mice expressing BCRs specific for hen egg lysozyme (HEL) along with soluble HEL (sHEL) as the cognate auto-Ag (IgTgsHEL mice; Goodnow et al., 1989; Hippen et al., 2000; Shlomchik, 2008). EndoU deficiency in IgTgsHEL mice also reversed AICD ex vivo and led to augmented anti-HEL auto-Ab responses in vivo. Thus, EndoU defines a new posttranscriptional regulatory pathway that controls B cell AICD, particularly in response to auto-Ag.
A genetic modifier locus/loci regulates BCR-induced AICD and CD5 expression
Spleen B cells from an inbred B6/129 founder line (CD22−/−[inbr]), their WT littermates (WT[inbr]), and WT B6 (WT[B6]) mice developed into blasts at normal frequencies and proliferated similarly after ex vivo BCR ligation using agonistic anti-IgM Abs (Fig. 1, A and B). In contrast, B cells from CD22−/− mice that were extensively backcrossed onto the B6 genetic background (CD22−/−[B6]) underwent AICD after BCR ligation. CD22−/−[B6] B cells also expressed CD5 after BCR stimulation but failed to up-regulate c-Myc transcript expression, whereas B cells from CD22−/−[inbr] had normal CD5 and c-Myc expression (Fig. 1, C and D). Similarly, B cells from IgTgsHEL mice with a B6 background underwent AICD, expressed CD5, and failed to up-regulate c-Myc after ex vivo BCR stimulation (Fig. 1, E–G). In contrast, B cells from IgTg mice (lacking the sHEL auto-Ag) blasted robustly, expressed CD5 at normal levels, and expressed high c-Myc levels after BCR ligation. These striking phenotypic similarities between CD22−/−[B6] and IgTgsHEL mice suggested that shared genetic and signaling programs regulated B cell AICD.
CD22−/−[inbr] mice were maintained as syngeneic brother/sister pairings for >8 yr, with genetic loci homozygous for either B6 germline DNA (B6:B6) or 129 germline DNA (129:129). To identify the genetic basis for the CD22−/−[B6] B cell phenotype, CD22−/−[B6] and CD22−/−[inbr] mice were first crossed to produce an F1 generation (CD22−/−[F1], Fig. 2 A) with either homozygous B6 (B6:B6) or heterozygous B6 and 129 (B6:129) genetic loci. CD22−/−[F1] B cell blast development after BCR stimulation was intermediate between B cells from CD22−/−[B6] and CD22−/−[inbr] mice (unpublished data), indicating that maximal AICD of CD22−/−[B6] B cells required B6:B6 homozygosity at a critical locus (or loci). CD22−/−[F1] and CD22−/−[B6] mice were subsequently crossed to generate genetically distinct CD22−/−[N1] littermates due to independent chromosomal segregation and crossovers between regions of heterozygous B6:129 genomic DNA during gametogenesis. Remarkably, B cells from CD22−/−[N1] mice underwent AICD (Fig. 2 B, top dot plots) or developed into blasts at substantial levels (Fig. 2 B, bottom dot plots), which correlated directly with increased or normal CD5 expression, respectively. These results confirmed the existence of a genetic modifier locus/loci that regulates BCR-induced AICD and CD5 expression.
A genetic locus regulates AICD of B cells from CD22−/−[B6] mice
Segregation of the B cell survival versus AICD phenotypes in CD22−/−[N1] mice indicated that the genomes of the parental CD22−/− mouse lines were stable and amenable to genetic analyses. Therefore, a forward genetic linkage analysis was used to identify the gene locus/loci responsible for CD22−/−[B6] B cell AICD. Genome-wide genotyping of 250 informative single nucleotide polymorphisms (SNPs) between the B6 and 129 mouse strains used genomic DNA from 44 CD22−/−[N1] mice; 22 mice had viable B cells after BCR ligation, whereas 22 mice had B cells that underwent AICD (Fig. 2 C). Quantitative trait loci (QTL) mapping regression analysis revealed a single locus with high logarithm of odds (LOD) scores (P < 10−6) on the distal end of chromosome (Chr) 15 (Fig. 2 D), with four consecutive SNPs spanning the region from 68–91 megabase pairs (Mbp, Fig. 2 E). LOD scores from the first three SNPs (68, 71, and 78 Mbp) were 8.3, increasing to 12.6 for the distal SNP (91 Mbp). The increased LOD score at 91 Mbp resulted from two mice (#14 and 28) with additional crossovers that were concordant with the observed phenotype. Parental CD22−/−[B6] mice were homozygous B6:B6 within the identified locus (68–91 Mbp), whereas CD22−/−[inbr] mice were homozygous 129:129. All other SNPs on Chr 15 outside of this locus (3.8–53 and 102 Mbp) were homozygous B6:B6 in all mice. Therefore, the primary genetic element responsible for CD22−/−[B6] B cell AICD was located within a 24 Mbp region.
To narrow the 78 to 102 Mbp Chr 15 locus, 23 additional SNPs within this region at 1 Mbp intervals were genotyped in the original 44 N1 mice. Using this method, the AICD phenotype mapped to the 94–98 Mbp region for all 44 CD22−/−[N1] mice (Fig. 2 F), including refined border regions for mice #14 and #28. Mice having apparently discordant phenotypes under low resolution mapping (viable, #2 and 19; nonviable, #44, Fig. 2 E) had concordant genotypes with higher resolution analysis (Fig. 2 F). To further refine and validate this locus as harboring the genetic element controlling AICD, additional SNPs and a total of 103 CD22−/−[N1] mice were analyzed (Fig. 2 G). Genotype and observed phenotypes were 98% (101/103) concordant in the mice, with additional crossovers observed in CD22−/−[N1] mice #21 and 64. Thus, the CD22−/−[N1] survival locus mapped to a refined 5 Mbp region spanning from 93.3 to 98.3 Mbp on Chr 15.
EndoU is overexpressed in B cells that undergo AICD
The 93.3–98.3 Mbp region encodes 17 pseudogenes and 38 known genes, including 10 olfactory receptor genes not expressed in hematopoietic cells. Therefore, the differential expression of transcripts from these genes was compared using BCR-stimulated CD22−/−[inbr] and CD22−/−[B6] B cells that remained similarly viable for 18 h. A single gene was differentially expressed between mouse lines, with 4.4-fold higher EndoU (formerly Pp11r/Tcl-30) levels in CD22−/−[B6] B cells compared with CD22−/−[inbr] B cells (Fig. 3 A). B cells from WT[B6] mice expressed EndoU at lower levels than B cells from WT 129 (WT) mice before and after BCR ligation (Fig. 3 B). EndoU transcript levels in CD22−/−[B6] B cells remained uniquely higher relative to CD22−/−[inbr] mice and all other genotypes after BCR ligation, whereas CD22−/−[F1] B cells expressed intermediate transcript levels. Thus, EndoU overexpression required CD22 deficiency in the context of a B6 genetic background.
To ensure that EndoU protein levels correlated with gene overexpression in CD22−/−[B6] B cells, an affinity-purified rabbit polyclonal Ab specific for recombinant EndoU was generated. Western blot analysis of whole cell lysates confirmed increased EndoU expression in both resting and BCR-stimulated CD22−/−[B6] B cells relative to CD22−/−[inbr] B cells (Fig. 3 C). EndoU overexpression in CD22−/−[B6] mice was B cell specific, as EndoU levels were modest in activated spleen T cells from CD22−/−[B6] and CD22−/−[inbr] mice. Remarkably, EndoU was also overexpressed in B cells from IgTgsHEL mice both before and after BCR ligation but was nearly undetectable in B cells from IgTg littermate mice (Fig. 3 D). Thus, B cell EndoU overexpression required both a homozygous B6:B6 EndoU locus in CD22−/−[B6] and IgTgsHEL mice that correlated with their characteristic AICD phenotypes.
Mammalian EndoU is a uridine-directed ssRNA binding protein
EndoU function in mammals is unknown, although EndoU transcripts are abundant in glucocorticoid-sensitive thymocytes undergoing apoptosis (Baughman et al., 1992). Highly conserved EndoU orthologs are found in many advanced and primitive species (see Fig. 8 A; Renzi et al., 2006), but EndoU paralogs derived from ancestral gene duplications do not exist in mice. The Xenopus ortholog of EndoU, XendoU, has been defined as an endonuclease targeting ssRNA molecules harboring polyuridine (poly(U)) regions (Renzi et al., 2006). Because EndoU substrates have not been defined in other species and EndoU-related molecules that could share redundant activities are not found in mice, the ability of EndoU to bind and/or process XendoU ssRNA substrates was examined. Recombinant EndoU protein specifically bound poly(U) ssRNAs, but not ssRNA species lacking these sites (Fig. 3 E). However, EndoU did not cleave these poly(U) ssRNAs, even in the presence of Mn2+, a condition favorable for XendoU endonuclease activity (Laneve et al., 2003). Mn2+ chelation did not interfere with EndoU binding of ssRNA molecules, as described for XendoU (Gioia et al., 2005). Thus, EndoU binds ssRNAs, likely through its predicted XendoU Superfamily domain (see Fig. 8 A).
Because c-Myc expression is reduced in CD22−/−[B6] and IgTgsHEL B cells after BCR ligation and c-Myc transcripts contain numerous stretches of poly(U) residues within the 3′ untranslated region (3′-UTR, Fig. 3 F), c-Myc mRNA interactions with EndoU were examined. Like the XendoU ssRNA substrate, c-Myc mRNA was also bound by recombinant EndoU (Fig. 3 F), but mRNA cleavage was not detectable (not depicted). Thus, c-Myc mRNA is a potential EndoU substrate, which may explain reduced c-Myc expression when B cells overexpress EndoU. Further supporting this notion, ectopic EndoU expression in mouse NIH-3T3 fibroblasts significantly reduced c-Myc protein levels (Fig. 3 G; 32% decrease, P < 0.05).
EndoU deficiency reverses CD22−/−[B6] B cell AICD
Because EndoU expression is low in WT and CD22−/−[inbr] B cells after BCR ligation (Fig. 3, B and C), the effect of reduced EndoU expression on CD22−/−[B6] B cell AICD was assessed. EndoU-deficient mice were generated by appropriately targeting the EndoU gene on B6 Chr 15 (Fig. 4, A–C). PCR-based SNP genotyping of genomic DNA from targeted mice confirmed that the targeted EndoU locus was B6 in origin. The chimeric mice were then bred onto a B6 genetic background to generate EndoU−/−[B6] mice that were homozygous B6:B6 throughout the 93.3–98.3 Mbp Chr 15 region (unpublished data). The absence of EndoU protein in homozygous gene-targeted mice was confirmed by Western blot analysis, which identified an appropriately sized protein in WT mice (Fig. 4 D). EndoU from CD22−/−[B6] B cells and thymocytes migrated more slowly on SDS-PAGE gels (∼50 kD band) than EndoU from CD22−/−[inbr] thymocytes (∼48 kD band, Figs. 3 C and 4 E), due to a nonsynonymous SNP present within EndoU exon 2 which switches a single Glu (B6 mice) to Lys (129 mice). This was verified using A/J mice that share the EndoU SNP sequence with 129 mice, with EndoU from both CD22−/−[inbr] and A/J mice migrating identically (Fig. 4 F). Splenocytes from CD22−/−[B6] and CD22−/−[inbr] mice expressed a single EndoU cDNA species as confirmed using 5′ RACE procedures (unpublished data). Thus, EndoU−/−[B6] mice do not express EndoU protein.
EndoU−/−[B6] and CD22−/−[B6] mice were crossed to generate double-deficient (EndoU−/−CD22−/−[B6]) progeny. A modest but significant reduction of total spleen B cell numbers is observed in CD22−/−[B6] mice, which normalized in EndoU−/−CD22−/−[B6] mice (Fig. 5 A). AICD was also reversed in B cells from EndoU−/−CD22−/−[B6] mice with normal BCR-induced blast development (Fig. 5 B) and proliferation (Fig. 5 C). c-Myc expression was also normalized in BCR-stimulated EndoU−/−CD22−/−[B6] B cells relative to CD22−/−[B6] B cells (Fig. 5, D and E). EndoU may preferentially regulate c-Myc expression in CD22−/−[B6] B cells after BCR ligation because proliferating cell nuclear Ag (PCNA) was expressed at normal levels (Fig. 5 E), as reported for other molecules critical for cell cycle progression (Poe et al., 2004). The rescue of EndoU−/−CD22−/−[B6] B cells from AICD appeared to be B cell intrinsic because lymphoid tissue and thymic development and cellularity were normal in EndoU−/− mice (unpublished data).
B cells from both CD22−/−[B6] and EndoU−/−CD22−/−[B6] mice shared a surface IgMlow phenotype (Fig. 5 F) that results from enhanced BCR tonic signals in the absence of CD22 negative regulation (Doody et al., 1995; O’Keefe et al., 1996; Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., 1997; Tedder et al., 1997). Otherwise, the modest reductions in surface IgD and CD21 expression that characterize CD22−/−[B6] B cells (Poe et al., 2004) were normalized in EndoU−/−CD22−/−[B6] B cells (Fig. 5 F). Enhanced heat stable Ag (HSA/CD24) expression is commonly used as a marker for immature/transitional B cells, but as with CD5, enhanced HSA expression is also characteristic of chronically stimulated or anergic B cells (Noorchashm et al., 1999). Regardless, the characteristic CD5highHSAhigh phenotype of CD22−/−[B6] B cells (Poe et al., 2004) was largely normalized in EndoU−/−CD22−/−[B6] B cells (Fig. 5 F), and CD22−/−[B6] mice have a normal ratio of mature to immature/transitional peripheral B cells (Poe et al., 2004). Thereby, elevated cell surface CD5 and HSA densities were phenotypic indicators for B cell AICD after BCR ligation.
EndoU-deficient IgTgsHEL mice produce auto-Abs
A defining feature of IgTgsHEL mice is the paucity of anti-HEL auto-Ab production. To examine EndoU function in this process, EndoU−/−IgTgsHEL mice were generated. Serum anti-HEL auto-Ab levels were strikingly high in some EndoU−/−IgTgsHEL mice, which were labeled “high responders” (Fig. 6 A). Anti-HEL auto-Ab levels in high-responder EndoU−/−IgTgsHEL mice were even greater than the levels of spontaneous anti-HEL Ab produced in IgTg mice (1.9-fold increase, P = 0.02) and EndoU−/−IgTg mice (1.8-fold increase, P < 0.01) lacking auto-Ag. High-responder EndoU−/−IgTgsHEL mice were present in both sexes, and carried both the IgTg and sHEL transgenes (Fig. 6 B). High serum anti-HEL auto-Ab levels developed in most high-responder mice after 5 wk of age and were maximal by 9–12 wk of age (Fig. 6, A and C). In addition to high-responder EndoU−/−IgTgsHEL mice, their age-matched littermates had augmented anti-HEL auto-Ab levels that were 2.4-fold higher than IgTgsHEL mice (P = 0.01, Fig. 6 A). Anti-HEL auto-Ab levels in augmented-responder mice continued to increase with age (Fig. 6 D), whereas anti-HEL auto-Abs were not enhanced in old IgTgsHEL mice (not depicted). Thus, EndoU overexpression regulates B cell auto-Ag responses in IgTgsHEL mice.
EndoU deficiency normalizes the phenotype of IgTgsHEL mice
Compared with IgTgsHEL mice, high-responder EndoU−/−IgTgsHEL mice had a marked increase in B cell blast development and c-Myc expression after BCR ligation, increased spleen B cell numbers and surface IgMa expression, and reduced CD5 and HSA expression (Fig. 6, E and F). In fact, high-responder mice were comparable to IgTg and EndoU−/−IgTg littermates lacking sHEL auto-Ag, except that surface IgMa levels were modestly decreased. In contrast, augmented-responder EndoU−/−IgTgsHEL mice remained phenotypically similar to IgTgsHEL mice. Some B cells in augmented-responder mice exhibited surface IgMa levels comparable to high-responder and IgTg B cells (Fig. 6 F, small peak), but otherwise their B cells had reduced IgMa expression, modest blast development and c-Myc expression after BCR ligation, and high CD5 and HSA expression, with low spleen B cell numbers (Fig. 6, E and F). The frequency of IgMa-high B cells in augmented-responder mice (11% ± 2.1%) was significantly greater than the frequency of IgMa-high B cells present in IgTgsHEL mice (2.2 ± 0.5%, P < 0.01). Thus, the normalized phenotype of high-responder EndoU−/−IgTgsHEL mice and the IgMa-lowCD5highHSAhigh phenotype of augmented-responder EndoU−/−IgTgsHEL B cells suggests differential sensitivities to auto-Ag exposure.
Whether reduced B cell AICD led to the depletion of sHEL auto-Ag in EndoU−/−IgTgsHEL mice with age was examined. Blood B220+ cells isolated from IgTg and high-responder EndoU−/−IgTgsHEL mice expressed relatively high surface IgMa levels and bound fluorescently labeled HEL protein at similar high levels (Fig. 7 A), suggesting that their BCRs were not occupied by sHEL. In contrast, B cells from both IgTgsHEL and augmented-responder EndoU−/−IgTgsHEL mice maintained an IgMa-low phenotype and did not bind labeled HEL protein, indicating BCR occupancy by sHEL. Dye-labeled spleen B cells from high-responder EndoU−/−IgTgsHEL mice were also adoptively transferred into WT or sHEL transgenic mice (Fig. 7 B). There was a dramatic reduction in IgM levels and HEL-binding capacity for high-responder B cells recovered from sHEL recipients in comparison with WT recipients (Fig. 7, B and C). Thus, the absence of AICD in high-responder EndoU−/−IgTgsHEL mice promoted auto-Ab production and endogenous sHEL clearance in vivo.
EndoU was identified in a gene modifier screen as a unique and potent regulator of B cell AICD. In WT[B6] mice, EndoU transcription and protein expression were suppressed by the balance between tonic BCR signals and their modulation by CD22, permitting normal BCR-mediated responses without blatant AICD after BCR ligation. In contrast, heightened BCR signals in peripheral B cells of CD22−/−[B6] mice drove EndoU overexpression and the CD5highHSAhigh phenotype with predominant AICD after BCR ligation. Genetic deletion of EndoU expression reversed the CD5highHSAhigh phenotype of B cells from CD22−/−[B6] mice and normalized AICD, confirming its important regulatory function in these processes. Bona fide EndoU substrates are likely to include c-Myc and other regulatory transcripts in vivo because c-Myc up-regulation is uniquely impaired in CD22−/−[B6] B cells (Poe et al., 2004), c-Myc expression was normalized in CD22−/−[B6] B cells by EndoU deficiency, EndoU bound c-Myc mRNA in vitro, and ectopic EndoU expression in a fibroblast cell line reduced c-Myc levels. A regulatory role for c-Myc in the mouse WEHI-231 B cell model of AICD has been previously described (Sonenshein, 1997; Donjerković and Scott, 2000). In CD22−/− mice, EndoU overexpression and AICD required B6 homozygosity at the EndoU locus. Therefore, genetic alterations must exist within the EndoU locus between the B6 and 129 genetic backgrounds that positively influences EndoU[B6] allele transcription in B cells, but not T cells. Although the EndoU locus has yet to be fine-mapped or sequenced in 129 mice, these important regulatory elements are likely to be located within the numerous stretches of conserved noncoding DNA present within the EndoU 5′ promoter region and 3′-UTR between B6 mice and humans (Fig. 8 C).
These collective observations support a model in which a CD22, EndoU, and c-Myc expression axis controls B cell fate after BCR ligation in B6 mice. EndoU levels remain low in WT[B6] B cells, allowing robust c-Myc expression and cell cycle progression after BCR engagement. In contrast, chronically high EndoU expression by CD22−/−[B6] B cells prevents c-Myc up-regulation after BCR ligation, resulting in AICD. EndoU was a major regulator of AICD in CD22−/−[B6] and IgTgsHEL mice, although additional unknown mechanisms undoubtedly also contribute to this complex regulatory process.
EndoU overexpression in vivo also occurred in response to BCR signals generated in IgTgsHEL mice, where B cells displayed a CD5highHSAhighIgMlow phenotype and failed to up-regulate c-Myc expression as was observed in CD22−/−[B6] mice. Genetic deletion of EndoU normalized this B cell phenotype in a substantial number of IgTgsHEL mice, which correlated with robust anti-HEL auto-Ab responses at an early age in mice that were defined as high responders. In augmented-responder EndoU−/−IgTgsHEL mice, some B cells expressed normalized phenotypes, suggesting that some B cells escape from an AICD phenotype with age to generate higher anti-HEL auto-Ab levels than IgTgsHEL mice. Given the linkage between EndoU, c-Myc up-regulation and AICD, it is likely that decreased B cell AICD in EndoU−/−IgTgsHEL mice leads to increased numbers of B cells that eventually consume endogenous sHEL auto-Ag, permitting clonal expansion with a resultant increase in anti-HEL auto-Ab production.
The divergence of phenotypes between high-responder and augmented-responder mice has precedent based on previous studies. For example, IgTgsHEL mice deficient in the inositol phosphatase PTEN have reduced BCR occupancy by sHEL and produce anti-HEL auto-Abs at a higher level than IgTgsHEL mice. This results in a subsequent break in tolerance in some mice caused by depletion of available sHEL auto-Ag (Browne et al., 2009). Likewise, IgTgsHEL mice transgenic for CD19 overexpression progressively break tolerance as they age and eventually produce high anti-HEL auto-Ab levels (Inaoki et al., 1997). In fact, remarkably similar to EndoU−/−IgTgsHEL mice, CD19TgIgTgsHEL mice develop as both high- and augmented-responder mice by 2 mo of age. Thus, modest increases in B cell numbers or enhancement in auto-Ab production resulting from altered B cell signaling can lead to auto-Ag depletion. Once this initial reduction in sHEL levels occurs, the resulting lack of B cell anergy enables peripheral B cell numbers to be maintained at a level that exceeds sHEL production, resulting in sustained and high-level anti-HEL auto-Ab production. Thereby, the EndoU regulatory pathway is likely to be an important component of B cell tolerance regulation, which is consistent with the known polygenic origins of autoimmunity.
The EndoU gene has an ancient origin, with conserved orthologs found in lower vertebrates (Suzuki et al., 2002; Gioia et al., 2005) and in invertebrates (Fig. 8, A and B). EndoU transcripts expressed in mouse B cells encoded a protein of 454 amino acids, which includes an N-terminal somatomedin B-like domain believed to be involved in protein–protein interactions (Gijsbers et al., 2003), a XendoU Superfamily ssRNA-binding domain, and a predicted transmembrane domain. EndoU specifically interacted with an ssRNA substrate harboring U-rich sequences that is also bound by XendoU, which is implicated in small nucleolar RNA processing in Xenopus (Gioia et al., 2005; Renzi et al., 2006). XendoU apparently generates snoRNAs through the cleavage of pre-mRNAs encoded within introns (Laneve et al., 2003). Human EndoU may exhibit low-level U-directed ssRNA endoribonuclease activity against the same in vitro ssRNA substrates recognized by XendoU (Laneve et al., 2008). However, mouse EndoU did not demonstrate measurable endonuclease activity against XendoU substrates. Mammalian EndoU may thus be functionally distinct from its Xenopus ortholog because human EndoU is excluded from the nucleolus of multiple cell lines (http://www.proteinatlas.org/ENSG00000111405/subcellular).
Viral orthologs of EndoU also exist (Fig. 8 B). Nsp15 (NendoU) is unique to the nidovirus family of ssRNA viruses, which includes the SARS coronavirus (Laneve et al., 2003; Bhardwaj et al., 2004; Ivanov et al., 2004; Gioia et al., 2005). Although Nsp15 is required for viral replication, specific Nsp15 substrates have not been identified and, surprisingly, appear to exclude the viral genome itself (Ivanov et al., 2004). Nsp15 has been postulated to suppress host immune responses (Ricagno et al., 2006) and to facilitate apoptosis in cells expressing the MAVS (mitochondrial anti-viral signaling) adapter protein that induces host anti-viral responses (Lei et al., 2009). Mouse hepatitis virus is also a member of the nidovirus family, is lymphotropic, and induces immunosuppression at least in part through the elimination of developing B cells (Lamontagne et al., 1989; Jolicoeur and Lamontagne, 1994). It therefore remains possible that mouse EndoU and hepatitis virus Nsp15 may target similar ssRNA substrates in B cells despite their evolutionary divergence. If true, each would have the capacity to suppress immune responses by inducing B cell AICD.
The current studies demonstrate for the first time that an RNA-binding protein plays a major role in regulating AICD, c-Myc expression, and the phenotype of peripheral B cells. Furthermore, these studies suggest an EndoU, CD22, c-Myc, and CD19 regulatory loop that directs normal B cell survival and malignant transformation (Fujimoto et al., 1999; Poe et al., 2012). EndoU likely regulates c-Myc and potentially other crucial ssRNA (mRNA or pre-mRNA) transcripts and nonclassical ssRNAs that directly regulate or fine-tune specific subsets of genes required for optimal B cell proliferation and survival. For autoreactive B cells that survive central selection and inhabit peripheral tissues, EndoU overexpression has nonetheless evolved as a checkpoint protein within the complex pathway that keeps potentially pathogenic B cells at bay through deletion via AICD.
MATERIALS AND METHODS
All procedures were approved by the Duke University Institutional Animal Care and Use Committee. CD22−/−[B6] and CD22−/−[inbr] mice were as previously described (Poe et al., 2004). EndoU−/−CD22−/−[B6] mice were produced by breeding EndoU−/− mice with CD22−/−[B6] mice, and then crossing heterozygous F1 animals to generate homozygous mice. WT littermates generated from the same F1 intercross served as controls. IgTg and IgTgsHEL mice (B6 background) were as previously described (Goodnow et al., 1989), with the IgTg and sHEL transgenes confirmed using JAX Mice Genotyping Protocols (The Jackson Laboratory).
To disrupt the EndoU gene by homologous recombination, an 8 kb DNA fragment containing exons 1–7 was isolated from a B6 genomic BAC clone (RP24-169P4). Flanking BsmBI restriction sites were used to remove the distal end of exon 4, a portion of intron 4 and the splice donor site, which was replaced with a neomycin-resistance gene (neor) that introduced an in-frame termination codon. A thymidine kinase (tk) gene was inserted at the 5′ end of the EndoU targeting sequence. Appropriate homologous recombination in EF1 (B6:129 hybrid) embryonic stem cells (Duke Transgenic Mouse Facility) was confirmed by Southern blot analysis of HindIII digested genomic DNA using a probe outside of the targeting sequence. The targeted allele generated an expected 8.5 kb band, compared with the WT 7.5 kb fragment. Correct EndoU gene targeting in subsequent offspring was verified by PCR analysis using a common forward primer (PCR1) in combination with reverse primers either 3′ of (PCR2) or within (PCR3) the inserted neor gene.
B cell blast development and proliferation.
Purified spleen B cells (B Cell isolation kit; Miltenyi Biotec) were analyzed by flow cytometry for blast development after 48 h of culture with F(ab′)2 goat anti–mouse IgM Abs as previously described (Poe et al., 2004). Alternatively, B cell proliferation during 72-h cultures was assessed by 3H-thymidine incorporation or CFSE labeling and dilution as previously described (Poe et al., 2004).
Flow cytometry analysis.
Assessment of cell surface Ags was performed by flow cytometry using fluorochrome-conjugated Abs as previously described (Poe et al., 2004). For intracellular c-Myc and PCNA expression, purified spleen B cells were cultured for 18 h in the presence or absence of F(ab′)2 anti–mouse IgM Ab and then fixed and permeabilized using a Cytofix/Cytoperm Plus kit (BD). Intracellular protein staining used either c-Myc Ab (IgG1 clone 9E10; BioLegend), followed by RPE-conjugated goat anti–mouse IgG1 secondary Abs (SouthernBiotech), or PCNA Ab (FITC-conjugated mouse IgG2a clone PC10; eBioscience). Immunofluorescence staining was assessed by flow cytometry, with background staining levels determined using nonreactive isotype-matched, fluorochrome-conjugated control Abs.
SNP genotyping and QTL analysis.
CD22−/−[B6] mice were crossed with CD22−/−[inbr] mice to generate syngeneic F1 progeny (CD22−/−[F1]). CD22−/−[F1] mice were backcrossed to CD22−/−[B6] mice to generate CD22−/−[N1] littermates. Blast development after BCR ligation was assessed for purified spleen B cells from CD22−/−[N1] littermates, along with B cells from single parental CD22−/−[B6] and CD22−/−[inbr] mice as controls. The results of 10 independent experiments were pooled. To reduce variability between mice and experiments, CD22−/−[N1] mice B cell blast frequencies >1 SD above the mean percentage blasts were considered viable, whereas B cell blast frequencies <1 SD below the mean were considered nonviable. Genomic DNA from 22 viable and 22 nonviable CD22−/−[N1] mice was used for genome-wide genotyping of 250 relevant SNPs between B6 and 129 mice (Illumina BeadArray; Duke University Genotyping Facility). QTL mapping used MapManager QTX regression analysis software (version QTXb20; Manly et al., 2001). Informative SNPs between B6 and 129 mice were identified using the Mouse Genome Informatics and dbSNP databases. DNA primer pairs were used to PCR amplify these SNPs and their surrounding sequences, which were sequenced (Duke Cancer Center DNA Analysis Facility) to identify single nucleotide (homozygous B6:B6) or double nucleotide (heterozygous B6:129) peaks at SNP sites.
Gene expression analysis.
Spleen B cells from mice of the indicated genotypes were cultured in triplicate for 18 h in medium alone or with F(ab′)2 anti–mouse IgM Ab, and then total RNA was isolated from pooled cells (RNeasy Plus; QIAGEN). For analysis of c-Myc and other signaling molecules, [32P]-radiolabeled cDNA was generated and hybridized to GEArray NF-κB signaling pathway gene arrays (SABiosciences) according to the manufacturer’s instructions, with hybridization assessed using x-ray film. Gene expression within the Chr 15 locus was analyzed using Mouse 430.2 RNA arrays (Affymetrix; Duke Cancer Inst. DNA Analysis Facility) with GeneSpring GX software (Agilent Technologies) analysis. For real-time PCR analysis, cDNA was synthesized using random primers and analyzed using a LightCycler Instrument (software version 3) and FastStart DNA MasterPLUS SYBR Green I kit (Roche Diagnostics Corp), with EndoU expression quantified using EndoU exon 5 (forward) 5′-CGTCAACGAGAAGCTGTTCTCCAAG-3′ and EndoU exon 6 (reverse) 5′-CCACATGTTCTTCAAATCGTCCAC-3′ primers. Cd20 expression was the internal control. Relative EndoU expression was quantified using the REST program (version 2).
Western blot analysis.
Polyclonal antiserum reactive with EndoU was generated using purified recombinant EndoU protein (347 amino acids, 107–454) fused at the C terminus with a 6-His tag and produced in the BL21 strain of E. coli. EndoU protein was affinity-purified on His-agarose, yielding a single band of correct size on SDS-PAGE gels. Rabbits were immunized and boosted with EndoU protein in adjuvant (Maine Biotechnology Services, Inc.). Anti-EndoU Ab was affinity-purified from immune sera using recombinant EndoU protein covalently bound to agarose beads. Purified spleen B cells or CD4+ T cells (CD4+ T Cell isolation kit; Miltenyi Biotec) were cultured with F(ab′)2 anti-IgM Abs for B cells or CD3e mAb for T cells (16.7 µg/ml, clone 500A2; BD) for 18 h. 106 cell equivalents of detergent lysates were separated by SDS-PAGE, followed by transfer to nitrocellulose membranes, with EndoU detected using anti-EndoU Ab, HRP-conjugated donkey anti–rabbit IgG (H+L) secondary Ab (Jackson ImmunoResearch Laboratories), and a Pico Enhanced Chemiluminescence kit (Pierce). The blots were stripped and reprobed with a polyclonal ERK2 Ab (C-14; Santa Cruz Biotechnology, Inc.) to ensure equivalent sample loading. Thymocytes (1.5 × 106 cell equivalents/lane) were also analyzed, with the blots reprobed using either polyclonal ERK2 Ab or polyclonal Lck Ab (2102, Santa Cruz Biotechnology, Inc.).
EndoU binding to RNA substrates and transfection of NIH-3T3 cells.
To assess EndoU binding activity, [32P]-labeled ssRNA substrates for XendoU (Laneve et al., 2008) were generated in vitro using dsDNA template oligonucleotides with the SP6 promoter to drive transcription. Full-length [32P]-labeled c-Myc mRNA was transcribed in vitro. Recombinant EndoU protein with a fused IgG binding domain tag was adsorbed onto MagnaBind goat IgG beads (Thermo Fisher Scientific), and incubated with substrate RNAs. Molar excess (100-fold) BSA and yeast tRNA were used as carriers. For XendoU substrates, the reactions were performed for 45 min in the presence or absence of 5 mM Mn2+ or 20 mM EDTA before analysis by 15% PAGE, with phosphorimager visualization. For c-Myc RNA, binding was assessed by scintillation counting.
To induce EndoU expression in mouse NIH-3T3 fibroblasts, cells were transfected (FuGENE HD; Promega) with a plasmid containing full length EndoU fused at the N terminus with CFP using the pECFP-N1 expression vector backbone (Takara Bio Inc.). Control-transfected reporter-positive NIH-3T3 cells were generated independently using the CFP (pECFP-N1) vector alone, or by co-transfection using the vector alone expressing GPF (pEGFP-C1), with similar results. Cells with stable plasmid incorporation were selected for appropriate reporter expression in the presence of 1 mg/ml Geneticin (Life Technologies).
Serum anti-HEL Ab levels in mice expressing the IgTg transgene were measured using serum diluted 1:100 in Tris-buffered saline containing 1% BSA as previously described (Inaoki et al., 1997).
Fluorescent labeling of HEL protein for flow cytometry analysis.
Fluorescently labeled HEL protein (Sigma-Aldrich) was made using Pacific blue succinimidyl ester (Invitrogen) according to the manufacturer’s instructions, and dialyzed 4× against a large volume of room temperature PBS.
Adoptive transfer experiments.
Purified spleen B cells (15 × 106) from high-responder EndoU−/−IgTgsHEL mice were labeled with eFluor 670 cell tracking dye (eBioscience) and injected intraperitoneally in sterile PBS into WT or sHEL recipient mice. After 36 h, peritoneal cavity and spleen leukocytes were harvested and analyzed by flow cytometry for the presence of the adoptively transferred B cells along with assessment of their IgMa surface expression levels and HEL-binding capacity.
We thank Drs. Bruce Beutler (Scripps Research Institute) and Jack Keene (Duke University) for advice, and Latoya McElreth and Isaac Sanford for technical assistance.
These studies were supported by National Institutes of Health grants AI057157, AI56363, and The Lymphoma Research Foundation.
The authors have no conflicting financial interests.
Author contributions: J.C. Poe, E.I. Kountikov, A. Natarajan, D.A. Marchuk, and T.F. Tedder conceived of and performed the experiments; J.C. Poe, E.I. Kountikov, D.A. Marchuk, J.M. Lykken, and T.F. Tedder analyzed the data; and all authors contributed to writing the manuscript.
3′ untranslated region
activation-induced cell death
hen egg lysozyme
heat stable Ag
logarithm of odds
proliferating cell nuclear Ag
quantitative trait locus
single nucleotide polymorphism
J.C. Poe and E.I. Kountikov contributed equally to this paper.