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Differences in immune responses to viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) can show sexual dimorphism. Age-associated B cells (ABC) are a population of CD11c+T-bet+ B cells critical for antiviral responses and autoimmune disorders. Absence of DEF6 and SWAP-70, two homologous guanine exchange factors, in double-knock-out (DKO) mice leads to a lupus-like syndrome in females marked by accumulation of ABCs. Here we demonstrate that DKO ABCs show sex-specific differences in cell number, upregulation of an ISG signature, and further differentiation. DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c− effector B cell populations with pathogenic and pro-inflammatory function as demonstrated by BCR sequencing and fate-mapping experiments. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs, resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function and differentiation of ABCs that accompanies TLR7-driven immunopathogenesis., Autoimmunity mediated by age-associated B cells (ABC) can affect males and females differently. Here, using a lupus-like mouse model that affects females more severely, the authors observe an ABC mediated and guanine nucleotide exchange factor (GEF) restrained pathogenic process involving TLR7.

Sex differences characterize immune responses to viruses and autoimmune diseases like SLE. ABCs are an emerging population of CD11c+ T-bet+ B cells critical for antiviral responses and autoimmune disorders. DEF6 and SWAP70, are two homologous molecules whose combined absence in double-knock-out mice (DKOs) leads to a lupus syndrome in females marked by an accumulation of ABCs. Here we demonstrate that DKO ABCs exhibit sex-specific differences in their expansion, upregulation of an ISG signature, and further differentiation. BCR sequencing and fate mapping experiments reveal that DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c- effector populations with pathogenic and proinflammatory potential. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function, and differentiation of ABCs contributing to the sex-bias that accompanies TLR7-driven immunopathogenesis.

Defects of filaggrin (FLG) compromise epidermal barrier function and represent an important known genetic risk factor for atopic dermatitis (AD), but also for systemic atopy, including allergic sensitization and asthma. The flaky tail mouse model, widely used to address mechanisms of atopy induction by barrier-defective skin, harbors two mutations that affect the skin barrier, the mutation Flgft, resulting in near-complete loss of FLG expression, and the matted mutation inactivating transmembrane protein 79 (Tmem79). Upon separation of the two mutant loci, which are closely linked on chromosome 3, mice defective only for Tmem79 featured pronounced dermatitis and systemic atopy. Upon extensive backcross to BALB/c, also Flgft/ft mice (assumed to be wild type for Tmem79), developed AD-like dermatitis and reproduced the human ‘atopic march’, with high IgE levels and spontaneous asthma, suggesting a key role for functional Flg in protection from atopy also in mice. In contrast, BALB/c mice congenic for a targeted Flg knock out mutation did not develop skin inflammation or atopy. To resolve this discrepancy, we generated Flg-deficient mice on a pure BALB/c background by inactivating the Flg gene in BALB/c embryos. These animals feature an ichthyosis phenotype, but do not develop spontaneous dermatitis or systemic atopy. We sequenced the genome of the atopic Flgft BALB/c congenics and discovered that they were unexpectedly homozygous for the atopy-causing Tmem79matted mutation. In summary, we show that Flg-deficiency does not cause atopy in mice. This finding is in line with lack of atopic disease in a fraction of Ichthyosis vulgaris patients carrying two FLG null alleles. However, absence of FLG may promote and modulate dermatitis caused by other genetic barrier defects, as skin inflammation in Tmem79ma/maFlgft/ft BALB/c congenics is qualitatively different compared to Tmem79ma/ma mice.

Loss of FLG causes ichthyosis vulgaris. Reduced FLG expression compromises epidermal barrier function and is associated with atopic dermatitis, allergy, and asthma. The flaky tail mouse harbors two mutations that affect the skin barrier, Flgft, resulting in hypomorphic FLG expression, and Tmem79ma, inactivating TMEM79. Mice defective only for TMEM79 featured dermatitis and systemic atopy, but also Flgft/ft BALB/c congenic mice developed eczema, high IgE, and spontaneous asthma, suggesting that FLG protects from atopy. In contrast, a targeted Flg-knockout mutation backcrossed to BALB/c did not result in dermatitis or atopy. To resolve this discrepancy, we generated FLG-deficient mice on pure BALB/c background by inactivating Flg in BALB/c embryos. These mice feature an ichthyosis phenotype, barrier defect, and facilitated percutaneous sensitization. However, they do not develop dermatitis or atopy. Whole-genome sequencing of the atopic Flgft BALB/c congenics revealed that they were homozygous for the atopy-causing Tmem79matted mutation. In summary, we show that FLG deficiency does not cause atopy in mice, in line with lack of atopic disease in a fraction of patients with ichthyosis vulgaris carrying two Flg null alleles. However, the absence of FLG likely promotes and modulates dermatitis caused by other genetic barrier defects.

Lipids affect the membrane properties determining essential biological processes. Earlier studies have suggested a role of switch-activated protein 70 (SWAP-70) in lipid raft formation of dendritic cells. We used lipidomics combined with genetic and biochemical assays to analyze the role of SWAP-70 in lipid dynamics. TLR activation using LPS as a ligand represented a pathogenic immunogenic stimulus, physical disruption of cell–cell contacts a tolerogenic stimulus. Physical disruption, but not LPS, caused an increase of phosphatidylcholine ether and cholesteryl esters in CD11c+ immune cells. An increase of ceramide (Cer) was a hallmark for LPS activation. SWAP-70 was required for regulating the increase and localization of Cers in the cell membrane. SWAP-70 controls Cer accumulation through the regulation of pH-dependent acid-sphingomyelinase activity and of RhoA-dependent transport of endosomal contents to the plasma membrane. Poor accumulation of Cers in Swap70−/− cells caused decreased apoptosis. This shows that two different pathways of activation, immunogenic and tolerogenic, induce different changes in the lipid composition of cultured CD11c+ cells, and highlights the important role of SWAP-70 in Cer dynamics in dendritic cells.

The nucleoporin ALADIN is required for multiple stages of oocyte maturation and for supporting embryonic divisions postfertilization., Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell’s center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.

Osteoclasts are bone resorbing cells acting as key mediators of bone disorders. Upon adhesion to bone, osteoclasts polarize and reorganize their cytoskeleton to generate a ring-like F-actin-rich structure, the sealing zone, wherein the osteoclast's resorptive organelle, the ruffled border, is formed. The dynamic self-organization of actin-rich adhesive structures, the podosomes, from clusters to belts is crucial for osteoclast-mediated bone degradation. Mice lacking the protein SWAP-70 display an osteopetrotic phenotype due to defective bone resorption caused by impaired actin ring formation in Swap-70−/− osteoclasts. To further elucidate the mechanisms underlying this defect, we investigated the specific function of SWAP-70 in the organization and dynamics of podosomes. These detailed studies show that the transition from podosome clusters to rings is impaired in Swap-70−/− osteoclasts. Live cell imaging of dynamic F-actin turnover and SWAP-70 localization during podosome patterning indicate that SWAP-70 is dispensable for cluster formation but plays a key role in F-actin ring generation. Our data provide insights in the role of SWAP-70's F-actin binding domain and pleckstrin homology (PH) domain in the proper localization of SWAP-70 and formation of a peripheral podosome belt, respectively. Ex vivo bone analyses revealed that SWAP-70-deficient osteoclasts exhibit defective ruffled border formation and V-ATPase expression. Our findings suggest an important role of membrane binding of SWAP-70 for the regulation of actin dynamics, which is essential for podosome patterning, and thus for the resorptive activity of osteoclasts., Highlights • SWAP-70 controls dynamic podosome patterning but not assembly of podosomes. • PIP3 and F-actin binding are required for proper subcellular localization of SWAP-70. • SWAP-70-deficient osteoclasts are impaired in ruffled border formation.

The journal of allergy and clinical immunology 144(4, Supplement) S31-S45 (2019). doi:10.1016/j.jaci.2019.02.004 special issue: "Mast cells in health and disease: summary of the German Research Foundation (DFG) priority programme : SPP 1394", Published by Elsevier, Amsterdam [u.a.]

Signal peptide peptidase (SPP) and the four homologous SPP‐like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II‐oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non‐expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail‐anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c −/− mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca 2+ ‐ATPase (SERCA2)‐regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca 2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c −/− mice.

Objective Treg cells need to acquire an effector phenotype to function in settings of inflammation. Whether effector Treg cells can limit disease severity in lupus is unknown. Interferon regulatory factor 4 (IRF-4) is an essential controller of effector Treg cells and regulates their ability to express interleukin-10 (IL-10). In non-Treg cells, IRF-4 activity is modulated by interactions with DEF-6 and its homolog switch-associated protein 70 (SWAP-70). Although mice lacking both DEF-6 and SWAP-70 (double-knockout [DKO] mice) develop lupus, they display normal survival, suggesting that in DKO mice, Treg cells can moderate disease development. The purpose of this study was to investigate whether Treg cells from DKO mice have an increased capacity to become effector Treg cells due to the ability of DEF-6 and SWAP-70 to restrain IRF-4 activity. Methods Treg cells were evaluated by fluorescence-activated cell sorting. The B lymphocyte-induced maturation protein 1 (BLIMP-1)/IL-10 axis was assessed by crossing DKO mice with BLIMP-1-YFP-10BiT dual-reporter mice. Deletion of IRF-4 in Treg cells from DKO mice was achieved by generating FoxP3(Cre) IRF-4(fl/fl) DKO mice. Results The concomitant absence of DEF-6 and SWAP-70 led to increased numbers of Treg cells, which acquired an effector phenotype in a cell-intrinsic manner. In addition, Treg cells from DKO mice exhibited enhanced expression of the BLIMP-1/IL-10 axis. Notably, DKO effector Treg cells survived and expanded as disease progressed. The accumulation of Treg cells from DKO mice was associated with the up-regulation of genes controlling autophagy. IRF-4 was required for the expansion and function of effector Treg cells from DKO mice. Conclusion This study revealed the existence of mechanisms that, by acting on IRF-4, can fine-tune the function and survival of effector Treg cells in lupus. These findings suggest that the existence of a powerful effector Treg cell compartment that successfully survives in an unfavorable inflammatory environment could limit disease development.

Post-transcriptional modifications can control protein abundance, but the extent to which these alterations contribute to the expression of T helper (TH) lineage-defining factors is unknown. Tight regulation of Bcl6 expression, an essential transcription factor for T follicular helper (TFH) cells, is critical as aberrant TFH cell expansion is associated with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here we show that lack of the SLE risk variant Def6 results in deregulation of Bcl6 protein synthesis in T cells as a result of enhanced activation of the mTORC1–4E-BP–eIF4E axis, secondary to aberrant assembly of a raptor–p62–TRAF6 complex. Proteomic analysis reveals that this pathway selectively controls the abundance of a subset of proteins. Rapamycin or raptor deletion ameliorates the aberrant TFH cell expansion in mice lacking Def6. Thus deregulation of mTORC1-dependent pathways controlling protein synthesis can result in T-cell dysfunction, indicating a mechanism by which mTORC1 can promote autoimmunity., Excessive expansion of the T follicular helper (TFH) cell pool is associated with autoimmune disease and Def6 has been identified as an SLE risk variant. Here the authors show that Def6 limits proliferation of TFH cells in mice via alteration of mTORC1 signaling and inhibition of Bcl6 expression.

Centromeres control genetic inheritance by directing chromosome segregation but are not genetically encoded themselves. Rather, centromeres are defined by nucleosomes containing CENP-A, a histone H3 variant [1]. In cycling somatic cells, centromere identity is maintained by an established cell cycle-coupled CENP-A chromatin assembly pathway, but how centromeres are inherited through the mammalian female germline is unclear because of the long (months to decades) prophase I arrest. We show that mouse oocytes retain the pool of CENP-A nucleosomes assembled before birth, and this pool is sufficient for centromere function, fertility, and genome transmission to embryos. Indeed, oocytes lack any measurable CENP-A nucleosome assembly through the entire fertile lifespan of the female (>1 year). Thus, the remarkable stability of CENP-A nucleosomes confers transgenerational centromere identity in mammals.

Mammalian meiocytes feature four meiosis-specific cohesin proteins in addition to ubiquitous ones, but the roles of the individual cohesin complexes are incompletely understood. To decipher the functions of the two meiosis-specific kleisins, REC8 or RAD21L, together with the only meiosis-specific SMC protein SMC1β, we generated Smc1βRec8 and Smc1βRad21L mouse mutants. Analysis of spermatocyte chromosomes revealed that besides SMC1β complexes, SMC1α/RAD21 and to a small extent SMC1α/REC8 contribute to chromosome axis length. Removal of SMC1β and RAD21L almost completely abolishes all chromosome axes. The sex chromosomes do not pair in single or double mutants, and autosomal synapsis is impaired in all mutants. Super resolution microscopy revealed synapsis-associated SYCP1 aberrantly deposited between sister chromatids and on single chromatids in Smc1βRad21L cells. All mutants show telomere length reduction and structural disruptions, while wild-type telomeres feature a circular TRF2 structure reminiscent of t-loops. There is no loss of centromeric cohesion in both double mutants at leptonema/early zygonema, indicating that, at least in the mutant backgrounds, an SMC1α/RAD21 complex provides centromeric cohesion at this early stage. Thus, in early prophase I the most prominent roles of the meiosis-specific cohesins are in axis-related features such as axis length, synapsis and telomere integrity rather than centromeric cohesion., This work was supported by grants fropm the Deutsche Forschungsgemeinschaft SPP1384 (RJ) and by the BFU (2014-59307-R) and JCyLe (AP).

The trend of women to become pregnant when older than in previous generations poses a paramount medical problem, for oocytes are particularly prone to chromosome missegregation, and aneuploidy increases with age. Recent data strongly suggest that as oocyte age increases sister chromatid cohesion is weakened or lost. Cohesin deterioration seems to contribute significantly to age-dependent aneuploidy, as discussed in this review.

Female mice lacking DEF6 and SWAP70 develop a lupus-like syndrome through dysregulation of IRF4 in activated B cells and plasma cells., Effective humoral responses to protein antigens require the precise execution of carefully timed differentiation programs in both T and B cell compartments. Disturbances in this process underlie the pathogenesis of many autoimmune disorders, including systemic lupus erythematosus (SLE). Interferon regulatory factor 4 (IRF4) is induced upon the activation of T and B cells and serves critical functions. In CD4+ T helper cells, IRF4 plays an essential role in the regulation of IL-21 production, whereas in B cells it controls class switch recombination and plasma cell differentiation. IRF4 function in T helper cells can be modulated by its interaction with regulatory protein DEF6, a molecule that shares a high degree of homology with only one other protein, SWAP-70. Here, we demonstrate that on a C57BL/6 background the absence of both DEF6 and SWAP-70 leads to the development of a lupus-like disease in female mice, marked by simultaneous deregulation of CD4+ T cell IL-21 production and increased IL-21 B cell responsiveness. We furthermore show that DEF6 and SWAP-70 are differentially used at distinct stages of B cell differentiation to selectively control the ability of IRF4 to regulate IL-21 responsiveness in a stage-specific manner. Collectively, these data provide novel insights into the mechanisms that normally couple and coordinately regulate T and B cell responses to ensure tight control of productive T–B cell interactions.

Eosinophils are the predominant inflammatory cells recruited to allergic airways. In this article, we show that human and murine eosinophils express SWAP-70, an intracellular RAC-binding signaling protein, and examine its role in mediating eosinophil trafficking and pulmonary recruitment in a murine model of allergic airway inflammation. Compared with wild-type eosinophils, SWAP-70–deficient (Swap-70−/−) eosinophils revealed altered adhesive interactions within inflamed postcapillary venules under conditions of blood flow by intravital microscopy, exhibiting enhanced slow rolling but decreased firm adhesion. In static adhesion assays, Swap-70−/− eosinophils adhered poorly to VCAM-1 and ICAM-1 and exhibited inefficient leading edge and uropod formation. Adherent Swap-70−/− eosinophils failed to translocate RAC1 to leading edges and displayed aberrant cell surface localization/distribution of α4 and Mac-1. Chemokine-induced migration of Swap-70−/− eosinophils was significantly decreased, correlating with reduced intracellular calcium levels, defective actin polymerization/depolymerization, and altered cytoskeletal rearrangement. In vivo, recruitment of eosinophils to the lungs of allergen-challenged Swap-70−/− mice, compared with wild-type mice, was significantly reduced, along with considerable attenuation of airway inflammation, indicated by diminished IL-5, IL-13, and TNF-α levels; reduced mucus secretion; and improved airway function. These findings suggest that regulation of eosinophil trafficking and migration by SWAP-70 is important for the development of eosinophilic inflammation after allergen exposure.

B-cell migration into and within lymphoid tissues is not only central to the humoral immune response but also for the development of malignancies and autoimmunity. We previously demonstrated that SWAP-70, an F-actin-binding, Rho GTPase-interacting protein strongly expressed in activated B cells, is necessary for normal B-cell migration in vivo. SWAP-70 regulates integrin-mediated adhesion and cell attachment. Here we show that upon B-cell activation, SWAP-70 is extensively posttranslationally modified and becomes tyrosine phosphorylated by SYK at position 517. This phosphorylation inhibits binding of SWAP-70 to F-actin. Phospho-site mutants of SWAP-70 disrupt B-cell polarization in a dominant-negative fashion in vitro and impair migration in vivo. After CXCL12 stimulation of B cells SYK becomes activated and SWAP-70 is phosphorylated in a SYK-dependent manner. Use of the highly specific SYK inhibitor BAY61-3606 showed SYK activity is necessary for normal chemotaxis and B-cell polarization in vitro and for entry of B cells into lymph nodes in vivo. These findings demonstrate a novel requirement for SYK in migration and polarization of naive recirculating B cells and show that SWAP-70 is an important target of SYK in this pathway.

Humans suffer a steep increase in aneuploidies when oocytes age, and deterioration of cohesin was suggested recently as a prominent cause. In the November 15, 2010, issue of Genes & Development, Tachibana-Konwalski and colleagues (pp. 2505–2516) answered a question central to this hypothesis: Can cohesin be reloaded onto mouse oocyte chromosomes long after birth? They found that it cannot, or at least not with an efficiency adequate to rescue cohesin deficiency. With no chance for sufficient replenishment, age-related loss of sister chromatid cohesion seems unavoidable.

Summary To ensure correct meiotic chromosome segregation, sister chromatid cohesion (SCC) needs to be maintained from its establishment in prophase I oocytes before birth until continuation of meiosis into metaphase II upon oocyte maturation in the adult. Aging human oocytes suffer a steep increase in chromosome missegregation and aneuploidy, which may be caused by loss of SCC through slow deterioration of cohesin [1–3]. This hypothesis assumes that cohesin expression in embryonic oocytes is sufficient to provide adequate long-term SCC. With increasing age, mouse oocytes deficient in the meiosis-specific cohesin SMC1β massively lose SCC and chiasmata [3, 4]. To test the deterioration hypothesis, we specifically and highly efficiently inactivated the mouse Smc1 β gene at the primordial follicle stage shortly after birth, when oocytes had just entered meiosis I dictyate arrest. In the adult, however, irrespective of oocyte age, chiasma positions and SCC are normal. Frequency and size of litters prove full fertility even in aged females. Thus, SMC1β cohesin needs only be expressed during prophase I prior to the primordial follicle stage to ensure SCC up to advanced age of mice.

Mast cells are effector cells of IgE-mediated immune responses frequently found at the vicinity of blood vessels, the margins of diverse tumors and at sites of potential infection and inflammation. Upon IgE-mediated stimulation, mast cells produce and secrete a broad spectrum of cytokines and other inflammatory mediators. Recent work identified JunB, a member of the AP-1 transcription factor family, as critical regulator of basal and induced expression of inflammatory mediators in fibroblasts and T cells. To study the impact of JunB on mast cell biology, we analyzed JunB-deficient mast cells. Mast cells lacking JunB display a normal in vivo maturation, and JunB-deficient bone marrow cells in vitro differentiated to mast cells show no alterations in proliferation or apoptosis. But these cells exhibit impaired IgE-mediated degranulation most likely due to diminished expression of SWAP-70, Synaptotagmin-1, and VAMP-8, and due to impaired influx of extracellular calcium. Moreover, JunB-deficient bone marrow mast cells display an altered cytokine expression profile in response to IgE stimulation. In line with these findings, the contribution of JunB-deficient mast cells to angiogenesis, as analyzed in an in vitro tube formation assay on matrigel, is severely impaired due to limiting amounts of synthesized and secreted vascular endothelial growth factor. Thus, JunB is a critical regulator of intrinsic mast cell functions including cross-talk with endothelial cells.

High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325-330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612-4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735-738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting approximately 5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR.

Cross-linking of the high-affinity IgE receptor (FcepsilonRI) on mast cell activates signaling pathways that trigger degranulation and the release of multiple pro-inflammatory mediators. Mature,immature and precursor mast cells are degranulation competent. We show here that the signaling protein SWAP-70 has a function in mast cell biology. While not found in many cell types, we find that apart from B cells, mast cells also express SWAP-70. In activated B cells, SWAP-70 shuttles between cytoplasm and nucleus, but in mast cells it is confined to the cytoplasm. SWAP-70(ko/ko) (double knockout) mice have reduced numbers of mature mast cells, and these are degranulation competent. However, although immature mast cells from SWAP-70(ko/ko) mice respond normally to SCF and IL-3 and have functional granules, their FcepsilonRI-mediated degranulation is inhibited. Thus, in mast cells SWAP-70 plays a role both in establishing the initial competence to degranulate and to develop into mature mast cells.

Tudor containing protein 6 (TDRD6) is a male germ line-specific protein essential for chromatoid body (ChB) structure, elongated spermatid development and male fertility. Here we show that in meiotic prophase I spermatocytes TDRD6 interacts with the key protein arginine methyl transferase PRMT5, which supports splicing. TDRD6 also associates with spliceosomal core protein SmB in the absence of RNA and in an arginine methylation dependent manner. In Tdrd6-/- diplotene spermatocytes PRMT5 association with SmB and arginine dimethylation of SmB are much reduced. TDRD6 deficiency impairs the assembly of spliceosomes, which feature 3.5-fold increased levels of U5 snRNPs. In the nucleus, these deficiencies in spliceosome maturation correlate with decreased numbers of SMN-positive bodies and Cajal bodies involved in nuclear snRNP maturation. Transcriptome analysis of TDRD6-deficient diplotene spermatocytes revealed high numbers of splicing defects such as aberrant usage of intron and exons as well as aberrant representation of splice junctions. Together, this study demonstrates a novel function of TDRD6 in spliceosome maturation and mRNA splicing in prophase I spermatocytes., Author summary Very little is known about splicing and its regulation in germ cells, particularly during meiosis. This paper describes the role of a male germ cell-specific protein, Tudor containing protein 6 (TDRD6), in assembly of the spliceosome in spermatocytes. We show that in prophase I TDRD6 interacts with the key protein methyltransferase of the splicing pathway PRMT5. PRMT5 methylates arginines in substrate proteins. In a methylation dependent manner, TDRD6 also associates with spliceosomal core protein SmB in the absence of RNA, thus before an RNP-type spliceosome has been assembled. In Tdrd6-/- diplotene spermatocytes PRMT5’s association with SmB and arginine dimethylation of SmB are much reduced. Abrogation of arginine methylation impairs the assembly of spliceosomes and the presence of the spliceosomal RNA U5 is aberrantly increased. These deficiencies in spliceosome maturation correlate with decreased numbers of Cajal bodies and gems involved in later stages, i.e. nuclear snRNP maturation. To reveal functional consequences of these deficiencies, transcriptome analysis of primary spermatocytes showed high numbers of splicing defects such as aberrant usage of intron and exons as well as aberrant representation of splice junctions upon TDRD6 loss. This study reveals a novel function of TDRD6 in spliceosome maturation and mRNA splicing in spermatocytes

The immunoglobulin heavy chain locus (Igh) features higher-order chromosomal interactions to facilitate stage-specific assembly of the Ig molecule. Cohesin, a ring-like protein complex required for sister chromatid cohesion, shapes chromosome architecture and chromatin interactions important for transcriptional regulation and often acts together with CTCF. Cohesin is likely involved in B cell activation and Ig class switch recombination. Hence, binding profiles of cohesin in resting mature murine splenic B lymphocytes and at two stages after cell activation were elucidated by chromatin immunoprecipitation and deep sequencing. Comparative genomic analysis revealed cohesin extensively changes its binding to transcriptional control elements after 48 h of stimulation with LPS/IL-4. Cohesin was clearly underrepresented at switch regions regardless of their activation status, suggesting that switch regions need to be cohesin-poor. Specific binding changes of cohesin at B-cell specific gene loci Pax5 and Blimp-1 indicate new cohesin-dependent regulatory pathways. Together with conserved cohesin/CTCF sites at the Igh 3′RR, a prominent cohesin/CTCF binding site was revealed near the 3′ end of Cα where PolII localizes to 3′ enhancers. Our study shows that cohesin likely regulates B cell activation and maturation, including Ig class switching.

The protein SWAP-70 was isolated as part of a DNA recombination complex in B lymphocytes, where it is predominantly expressed. In resting B cells, SWAP-70 is found in the cytoplasm; upon B-cell activation, it is transported both into the nucleus and to the cell membrane, where it is associated with the B-cell receptor complex and may play a role in signal transduction. In the nucleus, its involvement in heavy-chain class switch recombination has been suggested. In this report, using restriction fragment length polymorphism, simple sequence length polymorphism, and fluorescence in situ hybridization, we map the chromosomal localization of the mouse and the human genes to syntenic regions of mouse mid Chromosome (Chr) 7 and human Chr 11p15.

SWAP-70 is part of a protein complex that catalyzes cell-free DNA recombination between immunoglobulin heavy chain gene switch region substrates. This report studies the expression pattern of SWAP-70 in mouse tissues, sorted cells, and cultured primary cells. SWAP-70 RNA is strongly increased upon switch-induction of spleen cells, and very weakly expressed in thymus and bone marrow. SWAP-70 protein is specifically expressed in B cells, and levels increase rapidly after stimulation. Tissue staining shows strong expression in germinal center B cells, while macrophages and T lymphocytes do not stain. SWAP-70 is not detected in early B cells in the bone marrow. Its expression during mouse ontogeny after birth correlates with the appearance of non-IgM isotypes. While SWAP-70 localizes to the cell nucleus in activated B cells, it is not tightly associated with the chromatin and is found in the cytoplasm as well. SWAP-70 expression is not increased by gamma or UV irradiation of spleen cells, nor does it depend on p53. These characteristics are consistent with the putative role of SWAP-70 in immunoglobulin class switching.

SWAP-70 and DEF6, two proteins that feature similar domain and motif arrangements, are mainly known for their functions in differentiated hematopoietic cells. Both proteins interact with and regulate RhoGTPases and F-actin dynamics, yet their role in hematopoietic stem and precursor cells (HSPCs) remained unexplored. Here, the role of the SWEF proteins SWAP-70 and DEF6 in HSPCs was examined. Both SWEF proteins are expressed in HSCs. HSCs and different precursor populations were analyzed in mice deficient for SWAP-70, DEF6, SWAP-70 and DEF6 (double knockout, DKO), and wild-type controls. HSPCs isolated from these strains were used for competitive adoptive transfer into irradiated wild-type mice. Reconstitution of the myeloid and lymphoid lineages in the recipient mice was determined. The numbers of HSPCs in the bone marrow of Swap-70-/- and Swap-70-/-Def6-/- mice were >3-fold increased. When transplanted into lethally irradiated wild-type recipients, the reconstitution potential of Swap-70-/- HSPCs was intrinsically impaired in competing with wild-type HSPCs for contribution to hematopoiesis. Def6-/- HSPCs show wild type-like reconstitution potential under the same transplantation conditions. DKO HSPCs reconstituted to only 25% of wild-type levels, indicating a partial rescue by DEF6 deficiency in the Swap-70-/- background. Our study reveals the two SWEF proteins as important contributors to HSPC biology. Despite their similarity these two proteins regulate HSC/progenitor homeostasis, self-renewal, lineage contributions and repopulation in a distinct and mostly antagonistic manner.

The SCID (Severe Combined Immune Deficiency) mutation causes two DNA recombination deficiencies: an aberrant joining of V(D)J immunoglobulin gene elements and a failure to perform efficient repair of DNA double-strand breaks. A recently established cell-free assay for DNA transfer (DTA) was applied to study nuclear extracts from normal and SCID-derived cells. The recombination deficiency was reflected in the cell-free system: SCID lymphocyte and fibroblast extracts showed reduced levels of DTA activity on a variety of DNA substrates. Analysis of nuclear extracts prepared from wild-type thymocytes and B cells representing different stages in lymphocyte ontogeny revealed the highest activities at the most immature stages. With progression of development, DTA activity decreased. Corresponding to their early developmental arrest, V(D)J rearrangement-incompetent RAG-2-/- lymphocyte extracts show high DTA activity. In contrast, extracts from SCID early lymphocytes express very low DNA transfer activity. Induction of V(D)J rearrangement in vivo in a normal preB cell line lead to a co-induction of the cell-free recombination activity. This indicates a development stage specificity of cell-free DNA recombination, which temporally parallels V(D)J recombination. A protein could be purified to near-homogeneity from wild-type thymocytes which stimulates the recombination activity specifically in SCID thymocyte and proB cell extracts. This protein, SRSP (SCID Recombination Stimulatory Protein), migrates as a single band of approximately 72 kDa in SDS-polyacrylamide gel electrophoresis.

Bone remodeling involves tightly regulated bone-resorbing osteoclasts and bone-forming osteoblasts. Determining osteoclast function is central to understanding bone diseases such as osteoporosis and osteopetrosis. Here, we report a novel function of the F-actin binding and regulatory protein SWAP-70 in osteoclast biology. F-actin ring formation, cell morphology, and bone resorption are impaired in Swap-70(-/-) osteoclasts, whereas the expression of osteoclast differentiation markers induced in vitro by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) remains unaffected. Swap-70(-/-) mice develop osteopetrosis with increased bone mass, abnormally dense bone, and impaired osteoclast function. Ectopic expression of SWAP-70 in Swap-70(-/-) osteoclasts in vitro rescues their deficiencies in bone resorption and F-actin ring formation. Rescue requires a functional pleckstrin homology (PH) domain, known to support membrane localization of SWAP-70, and the F-actin binding domain. Transplantation of SWAP-70-proficient bone marrow into Swap-70(-/-) mice restores osteoclast resorption capacity in vivo. The identification of the role of SWAP-70 in promoting osteoclast function through modulating membrane-proximal F-actin rearrangements reveals a new pathway to control osteoclasts and bone homeostasis.

Background The regulation of normal and stress-induced erythropoiesis is incompletely understood. Integrin-dependent adhesion plays important roles in erythropoiesis, but how integrins are regulated during erythropoiesis remains largely unknown.Design and Methods To obtain novel insights into the regulation of erythropoiesis, we used cellular and molecular approaches to analyze the role of SWAP-70 and the control of integrins through SWAP-70. In addition, mice deficient for this protein were investigated under normal and erythropoietic stress conditions.Results We show that SWAP-70, a protein involved in cytoskeletal F-actin rearrangements and integrin regulation in mast cells, is expressed in hematopoietic stem cells and myeloid-erythroid precursors. Although Swap-70−/− mice are not anemic, erythroblastic differentiation is perturbed, and SWAP-70 is required for an efficient erythropoietic stress response to acute anemia and for erythropoietic recovery after bone marrow transplantation in irradiated mice. SWAP-70 deficiency impairs colony-forming unit erythroid development, while burst-forming unit erythroid development is normal, and significantly affects development of late erythroblasts in the spleen and bone marrow. The α4 integrin is constitutively hyper-activated in Swap-70−/− colony-forming unit erythroid cells, which hyper-adhere to fibronectin. Blocking α4 and β1 integrin chains in vivo restored erythroblastic differentiation and the erythropoietic stress response in Swap-70−/− mice.Conclusions Our study reveals that SWAP-70 is a novel regulator of integrin-mediated red blood cell development and stress-induced erythropoiesis.

The generation of plasma cells (PCs) is key for proper humoral immune responses. The transcription factors IRF-4 and BLIMP-1 (B-lymphocyte induce maturation protein-1) control PC commitment, but the underlying regulatory mechanisms are incompletely understood. Here we have identified SWAP-70 as being critically involved in Toll-like receptor (TLR)-triggered PC differentiation. Upon activation through various TLRs, Swap-70(-/-) B cells were activated and proliferated normally. However, expression of BLIMP-1 was markedly reduced and PC differentiation was impaired. Four hours of LPS stimulation were sufficient to drive PC differentiation, and SWAP-70 was required during this initial period. Swap-70(-/-) B cells pre-activated in vitro failed to efficiently differentiate into PCs upon adoptive transfer into recipient mice. Re-introduction of SWAP-70 into Swap-70(-/-) B cells rescued their development into PCs, and SWAP-70 over-expression in wild-type (WT) B cells increased PC generation. In the absence of SWAP-70, IRF-4 protein levels were reduced and the IRF-4(high) B220(+) CD138(-) compartment, including PC precursors, was strongly diminished. Ectopic expression of SWAP-70 increases IRF-4 protein levels and PC differentiation in WT and Swap-70(-/-) B cells, and IRF-4 over-expression in Swap-70(-/-) B cells elevates PC differentiation to WT levels. Thus, in a dose-dependent manner, SWAP-70 controls IRF-4 protein expression and thereby regulates the initiation of PC differentiation.

The term osteoimmunology is coined for molecular and cellular cross talk between the skeletal and immune system. Immunomodulatory signals have long been implicated as key regulators of bone metabolism. Recently, osteoclast-associated receptor (OSCAR), an IgG-like receptor, has been identified as an important osteoimmunological mediator. OSCAR expression in bone is highly conserved across different species, and the molecule is an important costimulatory receptor for osteoclast differentiation through activation of NFATc1. In humans, OSCAR is expressed by macrophages, monocytes, and monocyte-derived dendritic cells and modulates the response of the innate and adaptive immune systems by promoting cell activation and maturation, Ag presentation, and proinflammatory circuits. Human studies indicate that OSCAR may contribute to the pathogenesis and severity of osteoporosis and rheumatoid arthritis. In this paper, we review the structure-function relationship, expression pattern, and physiological role of OSCAR in osteoimmunology and summarize its potential implications for human diseases.

T1 and T2 transitional B cells are precursors for marginal zone B cells (MZB), which surround splenic follicles. MZB are essential for marginal zone formation, are central to the innate immune response, and contribute to adaptive immunity. Differentiation, migration, and homing of MZB and their precursors remain to be fully understood. We show that SWAP-70, a RhoGTPase-interacting and F-actin-binding protein with functions in cell polarization, migration, and adhesion regulates MZB development and marginal zone formation. The percentage of MZB in spleen of Swap70(-/-) mice was reduced to about one-third of that found in WT mice. Swap70(-/-) T1 cells accumulated in integrin ligand(high) regions of the splenic red pulp and failed to efficiently develop into T2 cells. Adoptive transfer and mixed BM chimera experiments demonstrated this to be a B-cell intrinsic phenotype. T-cell-independent antibody production was not impaired, however, and thus suggests that this process does not require correct homing of MZB precursors. B-cell adhesion through α(L)β(2) and α(4)β(1) integrins was hyper-activated in vitro and on tissue sections, and S1P-stimulated chemokinesis of MZB was reduced in the absence of SWAP-70. Thus, SWAP-70 acts as a regulator of the adhesion process, particularly important for differentiation control of B-cell precursors and their contribution to splenic tissue formation.

Osteoclasts are large, mobile, bone-resorbing cells and play a critical role in bone remodeling and catabolic bone diseases. The major function of osteoclasts is to hydrolyze inorganic hydroxyapatite and degrade organic bone matrix, mainly collagen. For evaluation of differentiation to fully functional osteoclasts in vitro, a quantitative functional resorption assay is essential. Currently available commercial test systems are either based on the organic or the inorganic part of the bone matrix. The novel resorption assay presented here is based on decellularized osteoblast-derived matrix. SaOS-2 cells were used for the synthesis of a densely mineralized extracellular bone matrix (ECM) in alpha-MEM medium, which strongly accelerates their matrix synthesis. After removal of the SaOS-2 cells, osteoclast precursors are plated on the osteoblast-derived matrix and stained by von Kossa. Subsequently, resorption pits were quantified by densitometry using an imaging program. Using this novel assay, we show that (i) RAW 264.7 cells resorbed the osteoblast-derived matrix continuously from day 6 until day 9 of culture, a process that is dose dependent on the macrophage colony-stimulating factor (M-CSF) concentration, (ii) the resorption performance of RAW 264.7 was dose-dependently inhibited by IFN-gamma, and (iii) the assay is working with primary human and mouse osteoclast precursors as well. In conclusion, this quantitative, functional, easy-to-use, inexpensive assay will advance analysis of osteoclast biology.

Cornelia de Lange syndrome (CdLS) is a clinically heterogeneous developmental disorder characterized by facial dysmorphia, upper limb malformations, growth and cognitive retardation. Mutations in the sister chromatid cohesion factor genes NIPBL, SMC1A and SMC3 are present in approximately 65% of CdLS patients. In addition to their canonical roles in chromosome segregation, the cohesin proteins are involved in other biological processes such as regulation of gene expression, DNA repair and maintenance of genome stability. To gain insights into the molecular basis of CdLS, we analyzed the affinity of mutated SMC1A and SMC3 hinge domains for DNA. Mutated hinge dimers bind DNA with higher affinity than wild-type proteins. SMC1A- and SMC3-mutated CdLS cell lines display genomic instability and sensitivity to ionizing radiation and interstrand crosslinking agents. We propose that SMC1A and SMC3 CdLS mutations affect the dynamic association between SMC proteins and DNA, providing new clues to the underlying molecular cause of CdLS.

To determine the site and mechanism of suppression by regulatory T (Treg) cells, we investigated their migration and function in an islet allograft model. Treg cells first migrated from blood to the inflamed allograft where they were essential for the suppression of alloimmunity. This process was dependent on the chemokine receptors CCR2, CCR4, and CCR5 and P- and E-selectin ligands. In the allograft, Treg cells were activated and subsequently migrated to the draining lymph nodes (dLNs) in a CCR2, CCR5, and CCR7 fashion; this movement was essential for optimal suppression. Treg cells inhibited dendritic cell migration in a TGF-beta and IL-10 dependent fashion and suppressed antigen-specific T effector cell migration, accumulation, and proliferation in dLNs and allografts. These results showed that sequential migration from blood to the target tissue and to dLNs is required for Treg cells to differentiate and execute fully their suppressive function. This work was supported by NIH R01 AI-44929, NIH R01 AI-62765, JDRF 1-2005-16, and the Emerald Foundation (all to JSB); NIH K08 AI071038 (to BS); and the Ministerio de Educación y Ciencia, Spain SAF2007-63579 (to JCO).

Mast cells, perhaps best known by their ability to trigger allergic reactions after stimulation through the FcepsilonRI, express the unusual phosphatidylinositol 3-kinase (PI3K)-dependent, Rac-binding protein SWAP-70. Here, we show that the IgE-mediated passive cutaneous and the systemic anaphylactic responses are strongly reduced in SWAP-70(-/-) mice. Cultured SWAP-70(-/-) immature bone marrow mast cells (BMMC) are also impaired in FcepsilonRI-mediated degranulation, which can be restored by expression of exogenous wild-type SWAP-70, but less so if a phosphatidylinositol trisphosphate (PIP(3)) binding mutant is expressed. SWAP-70 itself supports inositol-3-phosphate and PIP(3) production, the latter indicating a potential feedback from SWAP-70 towards PI3K. FcepsilonRI-stimulated transcription and release of cytokines is controlled by SWAP-70. Key FcepsilonRI signal transduction events like activation of LAT by phosphorylation, activation of Akt/PKB and of p38 MAP kinase are reduced in SWAP-70(-/-) BMMC, but ERK is strongly hyperactivated. Some requirements for SWAP-70 were apparent only under limited-strength signaling conditions. We suggest that SWAP-70 defines a new element of efficient mast cell activation upon FcepsilonRI signaling, important for the control of mast cell-dependent anaphylaxis.

Germinal centers (GCs) are lymphoid tissue structures central to the generation of long-lived, high-affinity, antibody-forming B cells. However, induction, maintenance, and regulation of GCs are not sufficiently understood. The F-actin–binding, Rac-interacting protein SWAP-70 is strongly expressed in activated B cells like those in B follicles. Recent work suggests that SWAP-70 is involved in B-cell activation, migration, and homing. Therefore, we investigated the role of SWAP-70 in the T-dependent immune response, in GC formation, and in differentiation into plasma and memory B cells. Compared with wt, sheep red blood cell (SRBC)–, or NP-KLH–immunized SWAP-70−/− mice have strongly reduced numbers of GCs and GC-specific B cells. However, SWAP-70−/− NP-specific B cells accumulate outside of the B follicles, and SWAP-70−/− mice show more plasma cells in the red pulp and in the bone marrow, and increased NP-specific Ig and antibody-forming B cells. Yet the memory response is impaired. Thus, SWAP-70 deficiency uncouples GC formation from T-dependent antibody and long-lived plasma cell production and causes extrafollicular generation of high-affinity plasma cells, but does not adequately support the memory response.

The migration of B cells into secondary lymphoid organs is required for the generation of an effective immune response. Here we analyzed the involvement of SWAP-70, a Rac-interacting protein involved in actin rearrangement, in B cell entry into lymph nodes. We noted reduced migration of Swap70-/- B cells into lymph nodes in vivo. Swap70-/- B cells rolled and adhered, yet accumulated in lymph node high endothelial venules. This defect was not due to impaired integrin expression or chemotaxis. Instead, Swap70-/- B cells aberrantly regulated integrin-mediated adhesion. During attachment, Swap70-/- B cells showed defective polarization and did not form uropods or stabilize lamellipodia at a defined region. Thus, SWAP-70 selectively regulates processes essential for B cell entry into lymph nodes.

The induction of alloantigen-specific unresponsiveness remains an elusive goal in organ transplantation. Here we identify plasmacytoid dendritic cells (pDCs) as phagocytic antigen-presenting cells essential for tolerance to vascularized cardiac allografts. Tolerizing pDCs acquired alloantigen in the allograft and then moved through the blood to home to peripheral lymph nodes. In the lymph node, alloantigen-presenting pDCs induced the generation of CCR4+ CD4+ CD25+ Foxp3+ regulatory T cells (Treg cells). Depletion of pDCs or prevention of pDC lymph node homing inhibited peripheral Treg cell development and tolerance induction, whereas adoptive transfer of tolerized pDCs induced Treg cell development and prolonged graft survival. Thus, alloantigen-presenting pDCs home to the lymph nodes in tolerogenic conditions, where they mediate alloantigen-specific Treg cell development and allograft tolerance.

Vectors based on the adeno-associated virus (AAV) have attracted much attention as potent gene-delivery vehicles, mainly because of the persistence of this non-pathogenic virus in the host cell and its sustainable therapeutic gene expression. However, virus infection can be accompanied by potentially mutagenic random vector integration into the genome. A novel approach to AAV-mediated gene therapy based on gene targeting through homologous recombination allows efficient, high-fidelity, non-mutagenic gene repair in a host cell.

Germinal centers (GC) are the sites of antigen-driven B cell switch recombination, V(D)J gene hypermutation, and selection to generate high-affinity CD38+ memory B cells. A marked expansion of GC associated with hypergammaglobulinemia followed by complete disruption of normal splenic architecture and a striking drop in immunoglobulin levels are prominent features of the murine retrovirus-induced immunodeficiency syndrome, MAIDS. B cell lymphomas are frequent in long-term infected mice. Normal GC formation is critically dependent on a number of genes including the transcription factor, Bcl6. Deregulated expression of BCL6 protein has been implicated in the development of human and mouse B cell lymphomas. Another nuclear protein, SWAP-70, has been identified as a subunit of the protein complex, SWAP, that recombines switch regions in vitro. To develop a fuller understanding of B cell biology in MAIDS, we examined the characteristics of BCL6, SWAP-70, CD38, and peanut agglutinin (PNA)-staining cells during the course of the disease. The levels of both nuclear proteins increased rapidly until 6–8 weeks after infection. During this time frame, BCL6 was expressed at highest levels in the usually rare CD4+ Thy1− T cell subset as well as in B cells. At later times, BCL6 levels dropped to undetectable levels while SWAP-70 levels continued to increase. Changes in the levels of either protein could not be ascribed to transcriptional regulation. PNA-reactive cells decreased in concert with BCL6 while CD38 staining increased with SWAP-70. These results demonstrate that progression of MAIDS results in the massive accumulation of B cells with the morphology of secretory cells that behave like post-GC cells for expression of BCL6 and CD38, and for PNA-staining but with abnormally high-level expression of SWAP-70.

Murine preB lymphocytes grow in tissue culture in the presence of stromal cells and interleukin 7 (IL-7), and can be induced to differentiate to surface-immunoglobulin-positive B cells in vitro by withdrawal of IL-7. Upon differentiation, proliferation ceases, and upregulation of Rag-1 and Rag-2 expression, and induction of V(D)J immunoglobulin-gene rearrangements occur. DNA-dependent protein kinase (DNA-PK) is required for effective V(D)J recombination and repair of DNA double-strand breaks. The holoenzyme comprises a catalytic subunit (DNA-PKcs) and the Ku heterodimer (Ku70/Ku80). We have analyzed expression of Ku70, Ku80 and DNA-PKcs upon induction of differentiation in preB cells derived from wild-type, severe combined immunodeficiency (SCID) and Rag-2−/− mice. Protein levels of Ku80 and Ku70 moderately decrease after induction in all three cell types. A distinct polypeptide that cross-reacts with anti-Ku Ig appears in the cytoplasm of wild-type and Rag-2−/− cells, but not of SCID cells. In mouse preB cells, Ku70 and Ku80 are present in the nuclei and cytoplasm before and after onset of differentiation. In vivo, Ku70 is predominantly expressed in V(D)J-recombination-active, early-preB and CD4−/CD8− thymocyte cell populations. Upon differentiation, protein levels of DNA-PKcs are unaltered. DNA-PK activity, which is not detectable in SCID cells, increases in wild-type and Rag-2−/− cells more than twofold shortly after induction of differentiation, then falls back to about 50% of starting levels.