Rap1 Signal Controls B Cell Receptor Repertoire and Generation of Self-Reactive B1a Cells

Immunity 24, 417–427, April 2006 ª2006 Elsevier Inc. DOI 10.1016/j.immuni.2006.02.007

Rap1 Signal Controls B Cell Receptor Repertoire and Generation of Self-Reactive B1a Cells Daisuke Ishida,1,4 Li Su,2,4,5 Akitoshi Tamura,1 Yoshinori Katayama,2 Yohei Kawai,1 Shu-Fang Wang,1 Masafumi Taniwaki,3 Yoko Hamazaki,2 Masakazu Hattori,1 and Nagahiro Minato1,2,* 1 Department of Immunology and Cell Biology Graduate School of Biostudies Kyoto University Kyoto 606-8501 Japan 2 Graduate School of Medicine Kyoto University Kyoto 606-8501 Japan 3 Department of Hematology Kyoto Prefectural University Kyoto 602-8566 Japan

Summary We previously reported that the mice deficient for SPA-1, a Rap1 GTPase-activating protein, developed hematopoietic stem cell disorders. Here, we demonstrate that SPA-12/2 mice show an age-dependent increase in B220high B1a cells producing anti-dsDNA antibody and lupus-like nephritis. SPA-12/2 peritoneal B1 cells revealed the altered Vk gene repertoire, including skewed Vk4 usage and the significant Igk/Igl isotype inclusion indicative of extensive receptor editing. Rap1GTP induced OcaB gene activation via p38MAPK-dependent Creb phosphorylation, and consistently, SPA-12/2 immature BM B cells showing high Rap1GTP exhibited the augmented expression of OcaB and Vk4 genes. SPA-12/2 BM cells could transfer the autoimmunity in association with the generation of peritoneal B220high B1a cells in Rag-22/2 recipients. Finally, a portion of SPA-12/2 mice developed B1 cell leukemia with hemolytic autoantibody. Present results suggest that the regulated Rap1 signal in the immature B cells plays a role in modifying the B cell receptor repertoire and in maintaining the self-tolerance. Introduction Rap1 (Ras-proximity-1) is a member of the Ras family of small G proteins ubiquitously expressed in most tissues and mediates broad cellular activities. The Rap1 signal plays an important role in controlling cell-cell and cellmatrix adhesion by regulating integrins and other adhesion molecules (Bos, 2005). It also regulates MAP kinases, including ERK and p38MAPK, depending on the cellular contexts and affects cell proliferation and survival, differentiation, and function (Stork, 2005). Rap1 is activated by a wide variety of stimuli, and this activa*Correspondence: [email protected] 4 These authors contributed equally to this work. 5 Present address: Center of Health Sciences, SRI International, Menlo Park, California 94025.

tion is mediated by specific guanine nucleotide exchange factors (GEFs), such as C3G, which is recruited by receptor-associated tyrosine kinases, and CalDAGGEF1 and Epacs, which are activated by Ca2+/diacylglycerol and cAMP, respectively (Bos, 2003). Rap1GTP, on the other hand, is inactivated by Rap1 GTPase-activating proteins (GAPs), which control the magnitude, duration, and localization of Rap1 signaling (Mochizuki et al., 2001). There are two groups of Rap1 GAPs, rapGAPs and SPA-1 family proteins, each of which bears unique protein interaction domains in addition to a conserved catalytic domain and shows a distinct tissue expression profile (Hattori and Minato, 2003). Recent studies have begun to uncover the roles of the Rap1 signal in the specific functional aspects of distinct tissue cell types. It is essential in embryonic development, and, thus, C3G deficiency results in the embryonic lethality that is partly due to the defective integrity of epithelial cells (Ohba et al., 2001). In neurons, the Rap1 signal is involved in the development of neuronal polarity as well as synaptic plasticity (Morozov et al., 2003; Pak et al., 2001; Schwamborn and Puschel, 2004), and, in renal tubular cells, the vasopression-regulated membrane translocation of aqaporin-2 may be controlled by Rap1 (Noda et al., 2004). The Rap1 signal plays multiple roles in hematopoietic cells (Kometani et al., 2004; Stork and Dillon, 2005). For instance, CalDAG GEF1 deficiency causes the insufficiency of platelet function leading to the bleeding diathesis (Crittenden et al., 2004), while SPA-1-deficient mice develop a spectrum of myeloprolifeartive stem cell disorders, including chronic myelogenous leukemia (CML) (Ishida et al., 2003a). Recent reports also indicate the significant roles of the Rap1 signal in controlling the invasion and metastasis of cancers (Park et al., 2005; Yajnik et al., 2003). Rap1 is a major activator of integrins in lymphocytes and plays an important role in the immunological synapse formation between T cells and antigen-presenting cells (Katagiri et al., 2002; Sebzda et al., 2002) as well as lymphocyte migration and trafficking (Katagiri et al., 2004). On the other hand, a series of reports indicate that Rap1 may mediate the negative signaling of T cell activation in certain conditions (Boussiotis et al., 1997; Katagiri et al., 2002). We showed that the increased Rap1GTP in the antigen-primed T cells of SPA-12/2 mice resulted in the anergic state due to the interference with T cell receptor (TCR)-mediated activation of the Ras/ERK pathway (Ishida et al., 2003b). More recently, it was reported that Rap1 played a role in the negative signaling in T cells via CTLA-4 as well as the anergic state of regulatory T cells (Dillon et al., 2005; Li et al., 2005). Although Rap1 is also activated strongly in B cells via B cell antigen receptor (BCR) stimulation (Christian et al., 2003), its role in the B lineage cells remains to be verified. In the present study, we show that SPA-12/2 mice develop lupus-like autoimmune disease characterized by an anti-dsDNA antibody and immune complex nephritis. We provide evidence that deregulated Rap1 activation in the immature BM B cells causes the altered IgL Vk

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Figure 1. Age-Dependent Increase in B1a Cells in SPA-12/2 Mice (A) Representative profiles of the two-color FACS analysis for the peritoneal cells from control and SPA-12/2 mice at various ages with the indicated antibodies. B1 cells are marked by rectangles (proportions are shown), and B2 cells are marked by circles. (B) Total numbers of the peritoneal B1 cells (B220+ Mac-1+), splenic follicular B cells (CD23high CD21low), and marginal zone (CD232 CD21high) B cells as well as the proportions of B1a cells (B220+ CD5+) in the peripheral blood lymphocytes. The means and SEs of the indicated numbers (n) of control (open columns) and SPA-12/2 (solid columns) mice of various ages are indicated.

gene repertoire and ineffective receptor editing, leading to the accumulation of self-reactive B1a cells in the peritoneal cavity, and we show that a portion of SPA-12/2 mice eventually develop B1 cell leukemia resembling human chronic lymphocytic leukemia (CLL). The results unveil a new aspect of the Rap1 signal linking B cell repertoire regulation, autoimmunity, and B1 cell leukemia.

SPA-12/2 mice showed strong anti-nuclear and/or cytoplasmic antibodies (Figure 2B). Histological examination revealed that the renal glomeruli in aged SPA-12/2 mice were enlarged and had increased mesangial cells, reduced Bowman’s capsule space, and deposition of IgM and C3, indicative of immune complex glomerulonephritis (Figure 2C).

Results

The Peritoneal B1 Cell Population in SPA-12/2 Mice Includes dsDNA-Reactive B Cells and Cycles In Vivo Freshly isolated peritoneal B1 cells of SPA-12/2 mice showed marked increase in Rap1GTP, whereas splenic B cells showed only a marginal increase (Figure 3A, left), suggesting the constitutive activation of the former in vivo. We therefore examined their cycling state in vivo by feeding the mice with BrdU for 2 weeks. While few peritoneal B1 cells incorporated BrdU in control mice, as much as 17% of those in SPA-12/2 mice were labeled with BrdU, indicating that a significant proportion of the SPA-12/2 B1 cells was cycling in vivo (Figure 3A, right). In the culture, SPA-12/2 B1 cells showed a rather reduced proliferation response to anti-IgM plus antiCD40 antibodies, but LPS-induced proliferation was unaffected, while SPA-12/2 spleen B cells proliferated in response to both stimuli comparably with the controls (Figure 3B). SPA-12/2 B1 cells apparently showed a higher threshold for BCR stimulation than control B1 cells, and they required roughly five times more antiIgM to attain the equivalent proliferation (Figure S3). Nonetheless, only SPA-12/2 B1cells showed significant proliferation in response to dsDNA antigen in vitro (Figure 3B). Also, SPA-12/2 B1 cells produced two times more IgM than control B1 cells by LPS stimulation, including significant anti-dsDNA antibody (Figure 3C). An elispot assay confirmed the presence of B cells producing anti-dsDNA IgG in the peritoneal cells of SPA-12/2 mice (data not shown). The results indicated

Age-Dependent Increase in the Peritoneal B220high B1a Cells Accompanied by Anti-dsDNA Antibody and Lupus-like Nephritis in SPA-1-Deficient Mice SPA-12/2 mice showed an age-dependent increase in the proportion of peritoneal B1 cells (IgMhigh B220+ CD232 CD21low), the majority of which exhibited CD5 expression, representing B1a cells (Figure 1A). It was noted that the B1a cells of aged SPA-12/2 mice showed homogenously higher intensity of B220 expression (B220high) than control B1a cells, while Mac-1 expression tended to be lower (Figure 1A), the former feature being shared with B1a cells of (NZBxNZW) F1 mice with lupus (Figure S1; see the Supplemental Data available with this article online). The total B1 cell numbers in SPA-12/2 mice were also increased in an age-dependent manner, whereas the numbers of both follicular (CD23high CD21low) and marginal zone (CD232 CD21high) B cells in the spleens remained comparable to the control littermates (Figure 1B). Significant B1a cells were also found in the circulation of SPA-12/2 mice (Figure 1B and Figure S2). The increased B1 cells showed no morphological features of transformation (data not shown). As shown in Figure 2A, SPA-12/2 mice showed significant IgM anti-dsDNA antibody at as early as 3 months of age, and many of them developed IgG, particularly IgG2b and IgG3, as well as IgA anti-dsDNA antibodies by 8 months of age. Also, about two-thirds of

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Figure 2. Development of Autoantibodies and Immune Complex Nephritis in SPA-12/2 Mice (A) Anti-dsDNA antibodies of various isotypes in the sera (3100 dilution) from control and SPA-12/2 mice at 3 and 8 months of age. The bars indicate the mean values. (B) Immunostaining of NIH3T3 cells (31000 magnification) with a control (wt) and SPA-12/2 mouse (KO) sera (200:1 dilution). (C) H&E staining (3400 magnification) and immunostaining with anti-IgM and anti-C3 antibodies (31000 magnification) of the kidneys from 8-month-old control and SPA-12/2 mice.

that the dsDNA-reactive B cell clones were accumulated preferentially in the peritoneal B1 cell population of SPA-12/2 mice, and they suggested that clones were activated by constitutive self-antigens in vivo despite the apparently elevated threshold for the BCR-mediated proliferation. Biased Vk Gene Repertoire and Ig Light Chain Isotypic Inclusion in the Peritoneal B1 Cells of SPA-12/2 Mice We then compared the BCR repertoires of B1 cells between SPA-12/2 and control mice by examining the Vk gene usage. A total of 202 and 102 Vk gene cDNAs were randomly cloned and sequenced from the purified peritoneal B1 cells of four SPA-12/2 and three control littermates of 8- to 10-month-old mice, respectively. Since individual mice showed similar tendency, the results were pooled. SPA-12/2 B1 cells showed a significantly altered overall Vk gene repertoire as compared with the control B1 cells, in which the frequency of Vk4 gene usage was markedly increased, reaching up to 40% (Figure 4, left). Unexpectedly, spleen B cells showed a largely similar Vk gene repertoire to the B1 cells in SPA-12/2 mice, while the repertoire between B1 and B2 cell populations differed significantly in control mice, as expected (Figure 4A, right). A distinct difference, however, was

found between the B1 and B2 populations of SPA-12/2 mice. Thus, as much as 15% of the SPA-12/2 B1 cells expressed both Igk and Igl chains, while few double expressers were detected in the splenic SPA-12/2 B cells (Figure 4B). Similar results were obtained in the three aged SPA-12/2 mice, the mean proportion of double expressers in the peritoneal B cells being 11.3%. It was noted that the Igk expression levels of double expressers were quite high (Figure 4B). In age-matched control mice, the double expressers were negligible in both B cell populations. The results strongly suggested that SPA-12/2 mice showed a significantly altered Vk gene repertoire shared by both B1 and B2 cell populations, while only the former was enriched with the B cells that had undergone apparently extensive receptor editing. The Rap1 Signal Activates OcaB Gene Expression via p38MAPK-Dependent Creb Phosphorylation We pursued the molecular linkage between the deregulated Rap1 signaling and the generation of self-reactive B1 cells by using a SPA-12/2 mouse-derived cell line, 1629B, which showed characteristic features of B1a cells (Figure S4A). While 1629B cells showed a high Rap1GTP level, restoring the SPA-1 gene expression abrogated the Rap1GTP accumulation. DNA microarray analysis revealed that a B cell-specific gene, OcaB, was

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scription. Rap1E63, however, failed to enhance the activity of a promoter bearing a mutation in the Creb binding site. Consistently, 1629B cells exhibited constitutive phosphorylation of Creb (Figure 5B, right). They also showed constitutive phosphorylation of both ERK and p38MAPK, and they showed that the inhibition of p38MAPK activation by a specific inhibitor without affecting ERK resulted in the suppression of both Creb phosphorylation and OcaB expression. The p38MAPK inhibitor barely affected the high Rap1GTP level, suggesting that Rap1was upstream of p38MAPK activation. These results strongly suggested that Rap1GTP promoted OcaB gene transcription via p38MAPKdependent Creb phosphorylation.

Figure 3. B1 Cells Are Responsible for Autoantibody Production and Cycle In Vivo in SPA-12/2 Mice (A) Rap1GTP in the peritoneal B1 and spleen B cells from 8-monthold control and SPA-12/2 mice was assessed by pull-down assay (left). Peritoneal cells from control (gray line) and SPA-12/2 (black line) mice fed with BrdU in their drinking water for 2 weeks were stained with anti-BrdU, B220, and CD5 antibodies, and the proportions of BrdU+ cells in the B1 cell fractions are indicated (right). (B and C) Peritoneal B1 and spleen B cells from 8-month-old control (open columns) and SPA-12/2 (solid columns) mice were cultured in the absence or presence of anti-IgM plus anti-CD40 antibodies (20 mg/ml each), LPS (100 mg/ml), or calf thymus dsDNA (100 mg/ml) for 48 hr, and the proliferation response was assayed. The means and SEs of four and seven mice, respectively, are shown. The horizontal line indicates the response in the medium alone. Supernatants of the cultures with or without LPS were harvested, and total as well as anti-dsDNA IgM were determined by ELISA.

most strongly overexpressed in 1629B compared with 1629B/SPA-1 cells (Figure S4B). The augmented OcaB gene expression in 1629B cells was confirmed by both Northern and immunoblotting (Figure 5A). The effect was further confirmed in the WEHI231 B cell line bearing a LoxP-flanked SPA-1 transgene, in that the induction of SPA-1 by Cre-mediated recombination abrogated the basal Rap1GTP and concomitantly suppressed the endogenous OcaB expression (Figure 5A). While constitutive IgM production was reduced in 1629B/SPA-1 cells, the forced overexpression of OcaB resulted in significant restoration (Figure 5A). A luciferase reporter assay indicated that the expression of Rap1E63 (an active form), but not Rap1N17 (an inactive form), enhanced the promoter activity of a basic regulatory region of the OcaB gene in both B (P3U1) and 293 cells, and the effect was abolished by the cotransfection of SPA-1 (Figure 5B, left). C3G, a Rap1GEF, also strongly enhanced the promoter activity, indicating that the activation of endogenous Rap1 promoted OcaB gene tran-

Augmented OcaB Expression in Peritoneal B1 Cells and BM Immature B Cells with Enhanced Vk4 Gene Expression of SPA-12/2 Mice We then examined the expression of OcaB in the primary B lineage cells of SPA-12/2 mice. Both spleen B and peritoneal B1 cells from control mice only marginally exhibited OcaB expression. In contrast, B1, but not spleen B, cells of SPA-12/2 mice showed constitutive phosphorylation of p38MAPK and Creb and markedly augmented expression of OcaB (Figure 5C, left) concomitantly with the high Rap1GTP level (see Figure 3A). RT-PCR analysis confirmed the increase in the OcaB transcript in SPA-12/2 B1 cells (Figure 5C, center). In the BM of SPA-12/2 mice, B lineage cells depleted of IgDhigh mature B cells showed the accumulation of Rap1GTP, and, again, this was associated with the enhanced phosphorylation of p38MAPK and Creb as well as the augmented OcaB expression (Figure 5C, right). SPA-12/2 mice showed increased overall B lineage cells (B220+), and this was mostly due to the increase in the immature (IgM+ IgDlow) and some transitional (T1) B cells (IgM+ CD212/low), with the total numbers of pro-/pre-B cells being unchanged (Figure 5D, left). Furthermore, RT-PCR analysis revealed that the immature SPA-12/2 BM B cells exhibited enhanced expression of Vk, particularly the Vk4 gene (Figure 5D, right). The results indicated that Rap1-mediated OcaB upregulation paralleled the increased BM immature B cells with a skewed Vk4 gene expression in SPA-12/2 mice. Transplantation of SPA-12/2 BM Cells Results in the Generation of Peritoneal B220high B1a Cells and Anti-dsDNA Antibody in Rag-22/2 Recipients To investigate whether the development of autoimmunity was an autonomous feature of the SPA-12/2 BM stem cells, we transplanted the BM cells, either total or lin2, from SPA-12/2 and control mice into sublethally irradiated Rag-22/2 mice. In the recipients of SPA-12/2 BM cells, irrespective of total or lin2 population, significant anti-dsDNA IgG was detected from 3 months of age onward, and 3 out of 5 recipients showed quite high-titered anti-dsDNA IgG by 5 months of age (Figure 6A, left). While the peripheral leukocyte numbers were slightly reduced in the recipients of SPA-12/2 BM (Figure 6A, right), no hematologic abnormality was detected (data not shown). In the peritoneal cavities of the control BM recipients, very few B1a cells were generated (Figure 6B), and this is consistent with the previous reports (Hayakawa and Hardy, 1988). On the other hand,

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Figure 4. Altered Vk Gene Repertoire and Igk/Igl Isotype Inclusion in the B1 Cells of SPA-12/2 Mice (A) Frequencies of the usage of Vk gene families in the peritoneal B1 cells and spleen B cells of SPA-12/2 (solid columns) and control (open columns) mice. A total of 202 and 102 Vk gene cDNA clones from the peritoneal B1 cells of four SPA-12/2 and three control mice, respectively, and 137 and 142 cDNA clones from the spleen B cells of three SPA-12/2 and three control mice, respectively, were sequenced. (B) Peritoneal and spleen cells from 12-month-old control and SPA-12/2 mice were analyzed with the indicated antibodies. Proportions of Igk+ Igl2 and Igk+ Igl+ cells are indicated. Similar results were obtained in the analysis of three mice each.

the recipients of SPA-12/2 BM, particularly those that developed high-titered anti-dsDNA IgG, revealed the significant generation of B1a cells in the peritoneal cavity; essentially all of these B1a cells were B220high and were similar to those in the donors (Figures 7B and 7C). The results strongly suggested that the generation of peritoneal B220high B1a cells and the development of anti-dsDNA IgG antibody were SPA-12/2 BM cell-autonomous. Development of B1 Cell Leukemia with Hemolytic Autoantibody Resembling Human B-CLL While SPA-12/2 mice began to develop a spectrum of hematologic disorders, including CML at ages over 12 months, a portion of them (9 out of a total of 62 leukemic mice) were found to show a marked increase in circulating B cells (Figure 7A). They were highly homogenous, medium-sized lymphoid cells with B220+ CD5+ Mac-1+ phenotypes typical for B1a cells (Figures 7B and 7C). Southern blotting analysis of Ig JH-gene could be done in three cases, and they were found to be oligo- or monoclonal (data not shown). These mice showed splenomegaly (the mean 430 mg versus 150 mg of the control mice) and frequent lymphadenopathy, reminiscent of human B-CLL. A portion of them revealed a marked increase in the highly blastic B1 cells throughout the peritoneal cavity, spleen, lymph nodes, and other vital organs, and the leukemia cells showed chromosomal translocations, suggesting blastic progression (Figure S5). A hallmark of human B-CLL is the development of autoantibodies, often causing autoimmune hemolytic anemia. As shown in Figure 7D, sera from six out of the nine mice showed significant hemolytic activity, whereas none of the control or SPA-12/2 mice with CML did. The results suggested that B1a cells in SPA-12/2 mice might be prone to leukemia.

Discussion We demonstrated here that mice deficient for SPA-1, a negative regulator of the Rap1 signal, developed the lupus-like autoimmune disease with occasional B1 cell leukemia resembling human B-CLL. It was suggested that the altered repertoire and ineffective receptor editing of Vk genes in the immature B cells underlie the disease, resulting in the generation of self-reactive B1 cells and their self-antigen-driven expansion.

Expansion of dsDNA-Reactive B1 Cells and Lupus-like Nephritis in SPA-1 Deficiency SPA-12/2 mice revealed a progressive increase in peritoneal B1a cells as they aged, and this was accompanied by the development of IgG anti-dsDNA and nuclear antibodies. Primary SPA-12/2 B1 cells showed markedly increased Rap1GTP, and a significant proportion of them incorporated BrdU in vivo, indicating that they were constitutively stimulated and cycling. Much higher expression levels of B220 in the SPA-12/2 B1a cells than control counterparts might be consistent with constitutive activation (Rice et al., 2005). The B1, but not spleen B, cells of SPA-12/2 mice showed specific proliferation in response to dsDNA and produced anti-dsDNA antibody by LPS stimulation in vitro. Interestingly, the SPA-12/2 B1 cells showed a rather elevated threshold for the BCR-mediated proliferation, while that for LPS was unaffected. BCR-mediated, but not LPS-induced, B cell proliferation was shown to depend crucially on the Ras/ERK pathway (Coughlin et al., 2005), and thus the effect was probably secondary to the high Rap1GTP levels, which interfered with Ras-mediated ERK activation (Ishida et al., 2003b). Nonetheless, present results strongly suggest the chronic expansion of self-reactive

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Figure 5. OcaB Gene Activation by Rap1 Signaling via p38MAPK-Dependent Creb Phosphorylation and Augmented OcaB Expression in the B1 and Immature BM B Cells of SPA-12/2 Mice (A) SPA-12/2 1629B cells infected with an empty MIG (1629B/GFP) or MIG-SPA-1 retrovirus (1629B/SPA-1) or WEHI231/loxP-SPA-1 B cells infected with an empty (Cre2) or Creadenovirus (Cre+) were examined for the expression of the OcaB mRNA by Northern blotting and the indicated proteins by immunoblotting. Rap1GTP was assessed by pulldown assay (left). 1629B/GFP (open columns), 1629B/SPA-1 (closed columns), and 1629B/SPA-1 cells further transduced with OcaB (gray columns) were cultured in the presence of PA6 stroma cells for the indicated periods, and IgM in the supernatants was determined by ELISA (right). (B) An OcaB promoter plasmid without (Wt) or with (Mu) mutation in the Creb binding site was cotransfected with an empty vector, pSRa containing Rap1E63, Rap1N17, SPA-1, or C3G cDNA into P3U1 myeloma or 293 cells. The doses of plasmids (mg) are indicated in parentheses. Means and SEs of the fold increases in luciferase activity normalized for the transfection efficiency of the three independent experiments are indicated (left). 1629B cells were cultured in the absence or presence of a p38MAPK inhibitor (SB202190) at the indicated concentrations, and the cell extracts were subjected to immunoblotting with the indicated antibodies or pull-down assay for Rap1GTP (right). (C) Lysates of the peritoneal B1 cells, spleen B cells, and BM B lineage cells depleted of the mature IgDhigh B cells from 10-monthold control and SPA-12/2 mice were immunoblotted with the indicated antibodies. RNAs from the aliquots of the B1 cells were subjected to RT-PCR analysis for OcaB and GAPDH. (D) BM cells from 12-month-old control and SPA-12/2 mice were multicolor analyzed by using the indicated antibody combinations. Proportions of total B cells (B220+), pro- and pre-B cells (IgM2 B220+) in the B220+ cell gate, immature B cells (IgM+ IgD2/low) in the B220+ cell gate, and transitional B cells (T1; IgM+ CD212 and T2; IgMhigh CD21+) in the lymphoid cell gate are indicated (left). RNAs from the aliquots of the immature BM B cells in (C) were subjected to RT-PCR analysis for the indicated genes (right).

B1a cells by stimulation with constitutive and abundant self-antigens in SPA-12/2 mice. Age-dependent increase in the peritoneal B1 cells has long been recognized in NZB and BWF1 mice (Hayakawa and Hardy, 1988). Their contribution to pathogenic autoantibody production and lupus-like nephritis, however, remains controversial (Atencio et al., 2004; Balabanian et al., 2003). Besides the natural lupus-prone mice, a number of gene-targeted mutant mice were also reported to develop anti-dsDNA/nuclear autoantibodies with or without lupus-like nephritis. In many of those, including CD222/2, FcgRIIB2/2, Aiolos2/2, and p21Cip12/2 mice, the autoimmunity was attributed to the general hyperactivity of the B and/or T cells (O’Keefe et al., 1999; Bolland and Ravetch, 2000; Sun et al., 2003; Balomenos et al., 2000). Recently, mice with a loss-offunction mutation in the RasGRP1 gene encoding a Ras activator were shown to develop lupus-like autoimmunity, in which autoantibody production was sec-

ondary to the autoreactive T cells that emerged through the defective thymic positive selection (Layer et al., 2003). Although the possible contribution of T cells to the autoimmunity in SPA-12/2 mice remains to be verified, our unpublished results indicate that the conditional expression of the Rap1E63 transgene in the B lineage cells also results in the development of anti-dsDNA antibody (Y.K. and N.M., unpublished data). Thus, SPA-12/2 mice provide a unique lupus model, in which the generation of self-reactive B1 cells and their self-antigen-driven expansion may play a primary role. Altered Vk Repertoire and ‘‘Partial’’ Receptor Editing in the Immature B Cells and Segregation of the Self-Reactive B Cells to the Peritoneal Cavity The B1 cell population in SPA-12/2 mice showed an altered Vk gene repertoire compared with the control counterpart, including a marked skewing for Vk4 gene usage, and, rather unexpectedly, the feature was shared

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Figure 6. Transfer of Autoimmunity into Rag22/2 Mice by SPA-12/2 BM Cells (A) BM cells, either total or lin2, of SPA-12/2 (solid circles) and age-matched control littermates (open circles) were transplanted to the 4.5Gy X-ray-irradiated Rag22/2 mice, and IgG anti-dsDNA in the sera was assessed at the indicated periods (left). Peripheral leukocyte counts of the control (open column) and the SPA-12/2 BM recipients (solid column) at 4 months after the transplantation are also indicated (right). (B and C) FACS profiles of the peritoneal cells of the control and SPA-12/2 BM recipients as well as the representative original donor mice. Total peritoneal cell numbers and proportions of the B220high B1a cells are indicated. Total numbers of the B220high B1a cells in the recipients of control (open circles) and SPA-12/2 (solid circles) BM were calculated. Double circles represent the recipients with high-titered IgG anti-dsDNA shown in (A).

with the SPA-12/2 spleen B cells. Because the immature BM B cells also showed enhanced expression of the Vk4 gene, the characteristic Vk gene repertoire involving both peripheral B cell populations was suggested to reflect the primary repertoire of the immature BM B cells. A

database analysis indicates that Vk4 is the most frequently utilized Vk gene among anti-dsDNA and ANA in mice (Liang et al., 2003), and, thus, enhanced Vk4 gene usage may favor the generation of immature B cells reactive to the nuclear autoantigens. It was also Figure 7. B1 Cell Leukemia with Hemolytic Autoantibody in SPA-12/2 Mice (A) SPA-12/2 mice with abnormal hemograms were followed carefully and sacrificed when they became moribund. Peripheral blood was analyzed for the T (open area), B (dark area), and myeloid (gray area) cells, and the proportions of each fraction in the total leukocytes are indicated. Means of ten control littermates are also shown. (B and C) Giemza staining (31000 magnification) and the FACS profiles of the blood leukocytes in a representative leukemic mouse (#3209). (D) Serially diluted sera from control, SPA-12/2 mice with myeloid leukemia, and SPA-12/2 mice with B1 leukemia were examined for the hemolytic activity.

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found that high proportions of the B1 cells, but not the spleen B cells, in aged SPA-12/2 mice were double expressers of IgL k and l chains, indicative of extensive receptor editing. It is indicated that a large proportion of the preselective BCR repertoire in normal immature B cells is self-reactive (Wardemann et al., 2003), and that the receptor editing at the k locus plays an important role in B cell tolerance for self-antigens (Chen et al., 1997; Retter and Nemazee, 1998). While successful edition of the self-reactive BCR by rare editor Vks negates self-reactivity, called ‘‘phenotypic exclusion,’’ rearrangement of Vk genes other than editor Vks may lead to repetitive rearrangement, which, in turn, eventually leads to the Vl gene rearrangement and expression called ‘‘partial editing’’ (Kenny et al., 2000; Li et al., 2001, 2002). Present results suggested that the SPA-12/2 immature B cells tended to show ‘‘partial’’ editing of the self-reactive BCR, generating the potentially self-reactive B cells. To support this, the transplantation of SPA-12/2 BM cells into Rag-22/2 mice resulted in the development of IgG anti-dsDNA antibodies concomitantly with the generation of B220high peritoneal B1a cells. The results suggested that the altered Vk gene repertoire and ‘‘partial’’ receptor editing in the immature B cells underlie the generation of self-reactive B cells in SPA-12/2 mice. In anti-dsDNA Ig transgenic mouse models, it was indicated that the ‘‘partially’’ edited, potentially pathogenic B cells expressing Igk/Igl dual receptors were segregated to the spleen marginal zone (Li et al., 2002). On the other hand, present results indicated that the self-reactive B cells in SPA-12/2 mice were segregated to the peritoneal cavity, although their presence in the marginal zone remained to be carefully examined. The exact mechanisms for the segregation of self-reactive B cells to the distinct privileged sites are unknown. It was reported that the marginal zone B cells of NZB mice exhibited enhanced b1-integrin expression (Atencio et al., 2004), and we found that the peritoneal B1a cells in SPA-12/2 mice also expressed much higher levels of b1-integrin than follicular B cells (D.I. et al., unpublished data). Since Rap1 controls the lymphocyte trafficking via integrin activation (Katagiri et al., 2004), it may be possible that the sustained Rap1 activation in the self-reactive SPA-12/2 B cells plays a role in their preferential homing to the peritoneal cavity. Although we found significant B1a cells in the circulation of aged SPA-12/2 mice, it remained to be investigated whether they represented the recirculating B1a cells or newly generated B1 cells homing to the peritoneal cavity. Upregulation of OcaB Gene Expression by the Deregulated Rap1 Signal in SPA-1 Deficiency: A Possible Role in Autoimmunity Present results revealed that the Rap1 signal potently activated OcaB gene expression via p38MAPK-dependent phosphorylation of its major transcription factor, Creb (Stevens et al., 2000). While Rap1-mediated activation of p38MAPK and possibly Creb has been reported in other cellular systems (Sawada et al., 2001; Schinelli et al., 2001), this is one of the first indications that the Rap1 signal mediates a gene activation specific for immune function. Consistently, the peritoneal B1 and BM immature B cells of SPA-12/2 mice, which showed

high levels of Rap1GTP, exhibited constitutive p38MAPK/Creb phosphorylation and augmented OcaB expression. OcaB is a B cell-specific transcriptional coactivator of Oct-1, 2 (Gstaiger et al., 1995; Luo and Roeder, 1995; Strubin et al., 1995), and the expression is tightly regulated during normal B cell development (Qin et al., 1998). OcaB plays crucial roles at multiple stages of B cell development, including early B cell development, terminal maturation of the recirculating B cells, and germinal center formation (Hess et al., 2001; Kim et al., 1996; Nielsen et al., 1996; Schubart et al., 1996). Most notably, OcaB is also crucial for the transcription and recombination of a selective subset of Vk genes, thus playing an important role in the modification of the BCR repertoire and receptor editing (Casellas et al., 2002). Among many Vk genes, Vk4 was reported to be one of the most OcaB-dependent genes and is highly editable in vivo when contributing to the selfreactive BCR (Casellas et al., 2001, 2002). The skewed expression of Vk in the immature SPA-12/2 B cells was consistent with the augmented expression of OcaB. We speculate that the excessive OcaB expression in the self-reactive immature B cells of SPA-12/2 mice promotes the ‘‘partial’’ receptor editing of the BCR by virtue of the selective preference for Vk genes other than editor Vks; this preference eventually leads to the isotype inclusion. The increase in the immature BM B cells in SPA-12/2 mice might, in part, reflect their prolonged sojourn at the stage of receptor editing (Casellas et al., 2001). This also raises an intriguing possibility that Rap1 may normally mediate a ‘‘self-sensing’’ signal via self-reactive BCR to initiate receptor editing via transient OcaB induction. Upregulated OcaB in the selfreactive B cells may also facilitate their survival (Hess et al., 2001). On the other hand, constitutive OcaB expression in the peripheral self-reactive B1 cells may contribute to enhanced autoantibody production and secretion. Interestingly, OcaB2/2 mice were also reported to develop autoantibodies, which was associated with the accelerated cell death of the transitional B cells (Jankovic and Nussenzweig, 2003). Thus, these results suggest that the tight regulation of OcaB gene expression by the Rap1 signal during B cell development may be crucial for maintaining B-cell self-tolerance. Possible Link between Autoimmunity and B1-Cell Leukemia via the Rap1 Signal It was found that a portion of SPA-12/2 mice eventually developed leukemia of B1 cell origin. While the majority of aged SPA-12/2 mice developed CML (Ishida et al., 2003a), B1 cell leukemia was unlikely to represent the lymphoid blast crisis of CML because it was not necessarily preceded by overt CML. B1 cell leukemia was frequently associated with the hemolytic autoantibody, reminiscent of human B-CLL, which is suspected to originate from B1 cells (Pangalis et al., 2002). Lupusprone mouse strains such as NZB/NZW and BWF1 also frequently develop B1-type CLL and hemolytic autoantibody (Phillips et al., 1992). It was reported that the transgenic mice of TCL-1 and APRIL genes showed a selective increase in the peritoneal B1a cells, followed by overt B-CLL, indicating that the aberrant activation of certain protooncogenes could cause B1 cell leukemia

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(Bichi et al., 2002; Planelles et al., 2004). In this aspect, it is noted that a number of potential protooncogenes are overexpressed in the SPA-12/2 B1 cell line, implying that the persistent Rap1 activation in the self-reactive B1 cells by constitutive self-antigen stimulation may predispose them to leukemia via activation of protooncogenes. Rap1 signaling thus may link autoimmunity and B-CLL. Experimental Procedures SPA-1 Gene-Targeted Mice SPA-12/2 mice (Ishida et al., 2003a) were backcrossed to C57Bl/6 mice over ten generations and routinely monitored by the hemogram and Giemza staining of blood films. SPA-12/2 mice with no hematological abnormalities were used for the analysis, while those with dubious hematological data were maintained until overt leukemia developed. C57Bl/6, BW (NZB 3 NZW) F1, and B6 Rag-22/2 mice were purchased from CLEA Japan, Inc., Japan, and all of the mice were maintained in an SPF condition at the Animal Center of the Kyoto University Medical School. Cytofluorimetric Analysis Cells were blocked with anti-FcgRIII antibody and stained with combinations of antibodies, followed by multicolor cytofluorimetric analysis with FACS Calibur (Beckton Dickinson). Antibodies used included APC-conjugated anti-B220, FITC-anti-Mac-1, PE-antiCD5, biotin-anti-CD5, PE-anti-CD23, PE-anti-IgD, FITC-anti-IgM (eBioScience), FITC-anti-Igl, PE-anti-Igk, FITC-anti-CD21, and APC-streptoavidin (BD Pharmingen). Cell Separations, Cell Cultures, and Lymphocyte Activation Assay Peritoneal B1 (B220+ CD5+) and spleen B (B200+) cells were isolated with a cell sorter (FACS Vantage, Beckton Dickinson). To enrich immature B cells, BM cells were incubated with the mixtures of PEconjugated anti-CD3, anti-Mac-1, anti-Gr-1, and anti-IgD, followed by anti-PE magnetic beads, and the negative fraction was collected by the AutoMax system (Meltenyl Biotech, Germany). The SPA-12/2 1629B cell line stably transfected with SPA-1 (1629B/SPA-1) or GFP alone (1629B/GFP) by using pMSCV-IRES/GFP (MIG) retroviral vector was cultured with PA6 stroma cells in complete BXH2 medium (Ishida et al., 2003a). 1629B/SPA-1 cells were further transduced with the OcaB gene by using a pMSCV-IRES/NGFR (MIN) retroviral vector containing Flag-tagged OcaB cDNA, and NGFR+ cells were sorted. WEHI231 cells were transfected with Flag-SPA-1 cDNA in a pCALNL expression vector, kindly provided by Dr. I. Saito, Tokyo University, Tokyo, Japan, by electroporation and were selected in G418-containing medium. To induce the SPA-1 expression, the cells were infected with Cre-containing adenovirus 2 days before use. For in vitro stimulation of lymphocytes, purified B cells were cultured in complete RPMI 1640 medium at 105 cells/well in the absence or presence of calf thymus double-stranded (ds) DNA (Sigma) at 100 mg/ml, anti-IgM plus anti-CD40 antibody at 20 mg/ml each, or LPS at 100 mg/ml for 48 hr and pulsed with 10 mCi 3H-thymidine followed by scintillation counting. BrdU Labeling In Vivo Mice were fed with BrdU (BD Bioscience) in their drinking water at 0.8 mg/ml for 2 weeks. Peritoneal B1 cells were fixed and permealized by BD Cytofix/Cytoperm buffer (BD Bioscience) for 30 min on ice, treated with 30 mg/ml DNase for 1 hr at 37ºC, and stained with FITC-conjugated anti-BUdR (BD Bioscience). Detection of Autoantibodies, Immunostaining, Karyotyping, and Cell Transfer Anti-dsDNA antibody was detected by using ELISA. Serially diluted sera or culture supernatants were incubated in the immunoplates (Nunc) precoated with 5 mg/ml cellulose-conjugated calf thymus dsDNA (Sigma) followed by alkaline phosphatase-conjugated antimouse Ig antibody specific for each isotype (Southern Bio Technology Associates). Hemolytic antibody was detected by incubating the serially diluted sera with a washed normal mouse RBC suspen-

sion in 96-well round-bottom plates in the presence of rabbit complement (Cedarlane Laboratories Ltd., Canada) for 1 hr. To detect antinuclear antibodies, NIH3T3 cells were fixed in 3% formaldehyde, permealized with 1% Triton X-100/PBS, blocked with 2% BSA, and incubated with diluted sera for 30 min, followed by FITC-anti-IgM antibody. Kidneys were either fixed in formaldehyde for hematoxylin and eosin (HE) staining or snap frozen in OCT for immunostaining with FITC-anti-IgM and anti-C3 antibodies (BD Bioscience). For cell transfer study, either total (2 3 107/mouse) or lin2 BM cells (5 3 105/mouse) were injected intravenously into 4.5 Gy X-ray-irradiated Rag22/2 mice. Pull-Down Assay and Immunoblotting Immunoblotting was done as before by using anti-SPA-1 (Tsukamoto et al., 1999), anti-Rap1, anti-Bob1 (OcaB) (Santa Cruz), antiphospho-Creb, anti-phospho-p38MAPK, and anti-phospho-ERK (Cell Signaling). Rap1GTP was detected by a RalGDS pull-down assay as described before (Tsukamoto et al., 1999). RT-PCR and Vk cDNA Cloning Total RNAs were extracted with ISOGEN Reagent (Nippon Gene, Toyama, Japan) and treated with DNaseI (Invitrogen BV, Carlsbad, CA). The cDNAs were synthesized with reverse transcriptase SuperScript III (Invitrogen BV) and amplified by using the Expand High Fidelity PCR System (Roche Diagnostics GmbH, Mannheim, Germany). Primer sequences were as follows: CD19, 50 -CTATGAGAAC GACTCCAACC-30 and 30 -CTAGGTCGTCAGACTTATCC-50 ; universal Vk, 50 -GGCTGCAGSTTCAGTGGCAGTGGRTCWGG-30 and 30 -GT CCTGATCAGTCCAACTGTTCAG-50 ; Vk4, 50 -CTGCMTCTCYDGG GGAGAAG-30 and 30 -CCWRMYCCACTGCCWMTRAAG-50 ; VpreB, 50 -CGTCTGTCCTGCTCATGCT-30 and 30 -ACGGCACAGTAATACA CAGCC-50 ; l5, 50 -CTTGAGGGTCAATGAAGCTCAGAAGA-30 and 30 -CTTGGGCTGACCTAGGATTG-50 ; OcaB, 50 -GGGAACATTATGT GCTGGCT-30 and 30 -GTCCTGAAGGACTGGCTCTG-50 . For Vk gene usage analysis, cDNAs from purified peritoneal B1 and splenic B cells were amplified with a universal Vk primer set, randomly cloned, and sequenced. Promoter Assay A 1.5 kb long promoter region of the OcaB gene (Stevens et al., 2000) was PCR amplified from murine genomic DNA and cloned into a pGEM-T easy vector. The primer set was 50 -GCCAGGAAGTTCA GGGGTTGAGTCAT-30 and 30 -TTTCCTCCCGTGGAACCACCG-50 . The promoter fragment was subcloned into SacI/XhoI sites of a pGVB2 vector. Mutation was introduced to CREB/ATF consensus binding sites (264 to 257 bp, GATGACGT to GATCTGGT; the mutation is underlined) by using a QuikChange Site-Directed Mutagenesis Kit (Stratagene). P3U1 or 293 cells were cotransfected with the OcaB reporter plasmid and various cDNAs in a pMX vector by using lipofectamineTM 2000 (Invitrogen). Cell extracts were harvested 24 hr after the transfection and were assayed for luciferase activity by using a Dual-Luciferase Reporter Assay System (Promega). The values of luciferase activity were normalized by cotransfection with the pRT-TK reporter plasmid. Supplemental Data Supplemental Data including five figures are available at http://www. immunity.com/cgi/content/full/24/4/417/DC1/. Acknowledgments This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan. Received: April 13, 2005 Revised: January 15, 2006 Accepted: February 1, 2006 Published: April 18, 2006 References Atencio, S., Amano, H., Izui, S., and Kotzin, B.L. (2004). Separation of the New Zealand Black genetic contribution to lupus from New

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