Cutting Edge: Absence of Expression of RAG1 in Peritoneal B-1 Cells Detected by Knocking into RAG1 Locus with Green Fluorescent This information is current as of September 26, 2021. Naomi Kuwata, Hideya Igarashi, Takafumi Ohmura, Shinichi Aizawa and Nobuo Sakaguchi J Immunol 1999; 163:6355-6359; ; http://www.jimmunol.org/content/163/12/6355 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ●

Cutting Edge: Absence of Expression of RAG1 in Peritoneal B-1 Cells Detected by Knocking into RAG1 Locus with Green Fluorescent Protein Gene1

Naomi Kuwata,*‡ Hideya Igarashi,* Takafumi Ohmura,* Shinichi Aizawa,† and Nobuo Sakaguchi2* Downloaded from based in the BM, B-1 cells maintain their numbers in adult animals It has been proposed that Ig gene rearrangement in the peri- by self-replenishment (4). The B-1 repertoire is fixed early in de- toneal cavity (Pc) B-1 cells might be involved in autoantibody velopment and becomes progressively restricted as animals age, generation. To study possible secondary maturation, we because new entrants to the B-1 pool are prevented (due to the feed prepared mice carrying a target integration of gfp gene into a back mechanism), and clonal populations expand to occupy a pro- ؉ rag1 locus (rag1/gfp mice). The GFP cells express rag1 mRNA gressively greater proportion of the pool. Many studies suggested and are undergoing Ig gene rearrangement. RAG1 expression that autoantibody-producing cells are generated from B-1 cells in http://www.jimmunol.org/ was studied in Pc B-1 cells to detect cells during the stage of Ig autoimmune-prone mice and in autoantibody-producing transgenic gene rearrangement. In contrast to previous reports, Pc B-1 (TG) mice (5, 6). cells did not show RAG1 expression in adolescent or elderly A recent paper demonstrated an increase of rag mRNAs in Pc mice. RAG1 expression was not induced in Pc B-1 cells in vivo B-1 cells from normal and the autoimmune New Zealand Black after stimulation by oral or i.p. administration of LPS. Our (NZB) mice (7). The results did not agree with the previous ob- results suggest that RAG1 expression in Pc B-1 cells is inhib- servation that B-1 cells possess a rather restricted range of Ab ited for a long period under normal condition and that this repertoire, that had been created before the recruitment from fetal

suppression is an essential state which maintains allelic exclu- liver or omentum of the Pc. The finding of the increased rag by guest on September 26, 2021 sion of Ig . The Journal of Immunology, 1999, 163: mRNAs in B-1 cells suggested a mechanism for secondary reper- 6355–6359. toire formation in the B-1 pool. The possibility of continuous Ig rearrangement could potentially be a major factor for leading to generation of autoantibody-producing B-1 cells. Therefore, we de- cells are generated from hematopoietic precursors by a signed experiments to study in vivo mechanisms of induction of specific molecular mechanism regulating rearrangements RAG1 reexpression in Pc B-1 cells. B of Ig genes (1, 2). Expression of recombinase compo- nents RAG1 and RAG2 is also regulated for the repertoire forma- Materials and Methods tion of a set of B cell clones generated in primary lymphoid organs. Targeting vector Reconstitution studies of various progenitor sources showed func- tionally distinct B cell populations as B-1 cells, derived from pro- A 9.0-kb rag1 fragment was subcloned into pBluescript II KS(Ϫ). EcoRI/ StuI fragment of rabbit ␤-globin poly(A) gene from pCXN2 (8) was ligated genitors that are present in fetal omentum and fetal liver but are 3 to EcoRI-StuI site of pEGFP (GFP). We prepared an EspI-CA-NcoI se- largely absent from adult bone marrow (BM) (3, 4). In contrast to quence into BamHI-EcoRI of vector, to which a NcoI-HindIII of pEGFP conventional B cells (termed B-2 cells), which are replenished poly(A) was ligated. A 1.3-kb EspI-ApaI GFP was ligated with endogenous throughout life by differentiation of unrearranged progenitors rag1 EspI and the ApaI of pBluescript II. The sequence (CAAC) from EspI to the ATG codon of rag1 was replaced with the sequence (CACC), giving a 1-nt difference in the 5Ј-flanking sequence. Departments of *Immunology, †Morphogenesis (Institute of Molecular Embryology and Genetics), and ‡Pediatrics, Kumamoto University School of Medicine, Honjo, Establishment of GFP knock-in mice Kumamoto, Japan Received for publication October 6, 1999. Accepted for publication October 25, 1999. TT2 were used for rag1/gfp knock-in embryonic stem (ES) cell (8). Genomic NcoI/BamHI-digested DNAs were hybridized with AM or 3UT The costs of publication of this article were defrayed in part by the payment of page (see Fig. 1A) from rag1 cDNA (1933–3115 bp and 3489–4706 bp). Mice charges. This article must therefore be hereby marked advertisement in accordance Ј with 18 U.S.C. Section 1734 solely to indicate this fact. were screened by PCR using RAG1-5-2; 5 -AGGTAGCTTAGCCAA- CATGG-3Ј, primer R6 and GFP3 (9). 1 The work was supported by the Ministry of Education, Science, and Culture of Japan, and by the NOVARTIS Foundation for the Promotion of Science. Flow cytometry 2 Address correspondence and reprint requests to Dr. Nobuo Sakaguchi, Department of Immunology, Kumamoto University School of Medicine, 2-2-1, Honjo, Kumamoto The mAbs are: PE-anti-mouse (m) B220 (RA3-6B2; PharMingen, San Di- 860-0811, Japan. E-mail address: [email protected] ego, CA), biotin-anti-mCD43 (S7), biotin-anti-mIgM (R6-60.2; PharMin- 3 Abbreviations used in this paper: BM, bone marrow; GFP, green fluorescent protein; gen), and biotin-anti-mCD5 (53-7.3; PharMingen). Streptavidin-RED670 Pc, peritoneal cavity; TG, transgenic; LM-PCR, ligation-mediated PCR. (Life Technologies, Rockville, MD) was used.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

● 6356 CUTTING EDGE

FIGURE 1. Generation of rag1/gfp knock-in mice. A, A targeting vector is com- pared with the rag1 locus and the homolo- gously recombined allele. DTA is a diphthe- ria toxin A gene for selection, AϩTisthe mRNA destabilizing sequence of G-CSF, and pau indicates a pausing signal of minute virus of mice (MVM) (8). The hatched box

is the RAG1 coding region. PCR was con- Downloaded from ducted with neo1, 5Ј-TCGTGCTTTACGG TATCGCCGCTCCCGATT-3Ј; and control 2, 5Ј-CTGGCCTCACTAAACGGCTCAG GCAATCTC-3Ј. B, Southern blot analysis of genomic DNA. DNAs were digested with NcoI and BamHI, and the hybridization was with AM or 3UT. The probes show bands at http://www.jimmunol.org/ 8.2 kb of the wild-type and 6.1 kb of the targeted allele. C, PCR of mouse tail DNA. Genomic DNA was amplified with primers of RAG1-5-2, R6, and GFP3. Normal and the targeting alleles were shown with either pair of RAG1-5-2 and R6 (515 bp) or RAG1-5-2 and GFP3 (626 bp). D, RT-PCR with BM RNAs were transcribed into cD- NAs and amplified with primers for rag1, gfp, and hprt. by guest on September 26, 2021

RNA and RT-PCR cell stage-dependent patterns in lymphoid lineage cells. Two lines B220ϩ cells by MACS beads were sorted into GFPϩ or GFPϪ cells on screened by PCR were confirmed by Southern blot analysis (Fig. FACSvantage. RNAs from sorted cells were analyzed by RT-PCR. The 1B). The 3UT and AM showed the adequate integration into rag1 cDNAs were amplified for rag1 with F3 and R6 (9), for gfp with F3 and gene as seen with the 6.1-kb band by NcoI/BamHI digestion. The GFP3; 5Ј-GCTCAGGTAGTGGTTGTCGG-3Ј, for hprt with HPRT5; and wild-type DNA showed rag1 gene on the 8.2-kb band. PCR 5Ј-GCTGGTGAAAAGGACCTC-3Ј and HPRT3. demonstrated the integration of gfp gene in the coding region of Ligation-mediated PCR (LM-PCR) rag1 gene (Fig. 1C). The endogenous 515-bp band was not de- tected in homozygous gfp/gfp mice, whereas heterozygous mice Genomic DNA was ligated to BW linker at 20 pmols (10) and PCR (11) showed both endogenous 515-bp and the targeted 626-bp bands. was carried out using the following primers: BW-1H and MuO2 for the first PCR; BW-1H and MuO for the nested PCR. The blot was hybridized with RT-PCR confirmed that homozygous gfp/gfp mice did not express the probe between primers. Control was with CD14 (10). rag1 mRNA but showed gfp mRNA instead (Fig. 1D), indicating that the mouse lines are rag1Ϫ but replaced with the gfp gene. Results and Discussion B220ϩ cells are composed of pro-B (G2; B220ϩCD43ϩ), pre-B Establishment of mice with the GFP knock-in to rag1 gene (G3; B220ϩCD43Ϫ), and immature or recirculating B cells (G4; The gfp knock-in to the coding region of one endogenous allele of B220highCD43Ϫ)(Fig. 2A). The BM of heterozygous rag1ϩ/gfpϩ the rag1 gene (Fig. 1A) will potentially represent results in ex- mice represents normal proportion of B cell development as G2 pression of both GFP and RAG1 in the identical tissue-specific and (20%), G3 (50%), and G4 (20%). GFPϩ cells are as 1.39% (G1), The Journal of Immunology 6357 Downloaded from http://www.jimmunol.org/

FIGURE 2. GFP expression as a marker for RAG1 and DNA breaks. A, GFP is shown in BM cells with expression of B220 and CD43. B220Ϫ cells by guest on September 26, 2021 do not express GFP. G3 expresses a higher level of GFP than G2. Half of G4 were GFPϪ. B, The rag1 and gfp mRNAs were confirmed. Total RNA isolated from GFPϩ cells of the BM or spleen was assessed by RT-PCR. Control HPRT increased in proportion to cycles. The populations of cells are: GFPϩB220ϩ BM cells (BMϩ), GFPϪB220ϩ BM cells (BMϪ), and GFPϪB220ϩ spleen cells (SPLϪ). C, LM-PCR for signal breaks in BM and spleen cells. DNA was purified from GFPϩB220ϩ BM cells (lane 1), GFPϪ BM cells (lane 2), GFPϪB220ϩ spleen cells (lane 3), wild-type mouse BM cells (lane 4), homozygous knock-in mouse BM cells (lane 5).

65.63% (G2), 89.68% (G3), and 55.25% (G4) (Fig. 2A). We next B-2 pool in the Pc. The GFPϩ cells in the B-2 population did not examined the capability in GFPϩ cells to mediate rearrangement of express transcripts of rag1 or gfp gene (data not shown), indicating Ig genes. B220ϩ cells of heterozygous rag1ϩ/gfpϩ mice were sep- that the signal of RAG1/GFP is more sensitive to chase newly arated by GFP as BMϩ and BMϪ. The endogenous rag1 mRNA generated B cells than the measurement by RT-PCR. There are no was detected abundantly in GFPϩ cells (Fig. 2B). Recombinase RAG1/GFPϩ cells (Ͻ1.5%) in the B-1 cells. This is consistent activity was measured by LM-PCR of GFPϩ and GFPϪ cells (Fig. with the idea that the B-1 pool was formed presumably when the 2C). PCR products indicating the occurrence of DJ rearrangement mice were neonates earlier stage and has been maintained for the were detected in GFPϩ cells at various sizes. BMϪ cells and longer period without rag1 gene expression in the Pc for a length GFPϪB220ϩ spleen cells did not show any signal. BM cells from of time sufficient for GFP protein to decay below the limits of wild-type littermate showed a single band of DJ rearrangement but detection. The result that very few B-1 cells showed RAG1/GFP the homologously replaced (gfpϩ/gfpϩ) mice showed no rear- expression is surprising because a recent report conversely dem- rangements. These results indicate that the GFP expression is reg- onstrated an increase of rag1 mRNA in Id negative B-1 cells of ulated under the endogenous rag1 promoter, suggesting that GFPϩ mice carrying replacement of IgVH and IgVL (7). We used the cells are undergoing Ig gene rearrangement. same mAbs to gate the B-1 cells and the cells also showed CD5, indicating that the Pc B-1 cells from 6-wk-old mice showed a very Pc B-1 cells do not express RAG1 signal in normal mice low level of RAG1 under special pathogen free conditions (Fig. We studied the expression of RAG1 in B-1 cells. Pc B cells are 3A). The B-1 cell is maintained its pool by self-replenishment with composed of two types: B220lowIgMhighCD5ϩ (B-1a or B-1) cells the reduced recombination activity in the Pc. and B220highIgMlowCD5Ϫ (B-2) cells. RAG1/GFP is detected in It is possible that B-1 cells from older mice may show the in- B-2 cells (Fig. 3A). The B-2 cells are presumably continuously duction of RAG1 during aging of the B-1 pool either by the re- supplied with newly rearranged B cell immigrants from the BM as population of newly generated B-1 cells or by activation of sec- that are detected before the GFP protein decays below detectable ondary Ig gene rearrangement as observed in the germinal center levels. These freshly repopulating cells make up ϳ15–45% of the (GC) region (9, 12). Therefore, we evaluated the effect of aging on 6358 CUTTING EDGE

FIGURE 3. GFP expression in B-1 or B-2 cells in the Pc. A, Pc cells were isolated from adolescent Downloaded from mice (4–8 wk) and stained with mAbs against B220, IgM, and CD5. B-1 and B-2 cells were gated with expression of IgMhighB220low or CD5ϩB220low as B-1 cells, and IgMlowB220high or CD5ϪB220high as B-2 cells. GFP of gated cells is shown and the num- ber indicates the percentage of GFPϩ cells. B, GFPϩ cells in Pc B-1 and B-2 cells from adolescent or el- http://www.jimmunol.org/ derly mice are shown. by guest on September 26, 2021

the regulation of RAG1/GFP expression in B-1 cells. The Pc B-1 gest that the Pc B-1 cell pool that is maintained by self-replenish- cells from 6-mo-old mice did not show any induction of RAG1/ ment is presumably stable in the Ab specificity because Ig gene GFP (Fig. 3B). While RAG1/GFP expression in the B-2 pool is recombination is suppressed in normal mice. detected as 33.15% in the adolescent mice, the RAG1/GFP ex- A similar chase study of RAG2 expression in mature B cells pression is absent in Pc B-1 cells of both adolescent and elderly using gfp-TG and knock-in mice was reported recently (14–16). mice. RT-PCR detected neither rag1 nor transcripts in the The mice showed that RAG2 was observed at the lower level in B-1 cells (data not shown). Administration of LPS activates Pc B-1 immature B cells of the spleen but an antigenic challenge would cells and causes the induction of autoantibody production or ex- not easily induce reexpression of RAG2. We also examined acerbation of autoimmune symptoms in the TG mouse model (13). RAG1/GFP expression after stimulating B-1 cells in vitro with Oral administration of LPS caused an increase of Pc cells in ϳ56% various B cell activators, but none of the attempt was successful of the control mice (from 3.20 to 4.98); however, we did not detect for induction of RAG1 at the detectable level (data not shown). any increase of RAG1/GFP expression in B-1 cells (Table I). We These results might not agree with recent observations of frequent further examined the effect of LPS on RAG1/GFP expression by expression of RAG and the induction of secondary Ig gene rear- direct administration into the Pc. Stimulation with LPS did not rangements in the germinal center by stimulation with T-depen- induce reexpression of RAG1/GFP in Pc B-1 cells in vivo, al- dent Ag (9, 12). though we detected a slight increase of GFPϩ cells in B-2 popu- The rag1/gfp knock-in mice showed that a single copy rag1 lation (from 17.48 to 20.04% by oral administration of LPS and promoter mediated expression of GFP that is sufficient to mark the from 10.85 to 12.16% by i.p. injection in vivo). These results sug- RAG1 expressing B-1 cells in vivo. Our results provide important The Journal of Immunology 6359

Table I. Effect of administration of LPS in vivo upon expression of RAG1/GFP in peritoneal B-1 and B-2 cellsa

GFP Positive Cells (%) in Route of No. of Administration No. of Cellsb B-1 B-2 Mice

Oral PBS 3.20 Ϯ 0.62 1.78 Ϯ 1.29 17.48 Ϯ 4.75 5 LPS 4.98 Ϯ 1.03 1.42 Ϯ 1.07 20.04 Ϯ 8.39 5

i.p. PBS 2.00 2.28 10.85 2 LPS 4.00 1.00 Ϯ 0.48 12.16 Ϯ 1.91 3

a Oral and i.p. administration of LPS was carried out as described (13), and the effect was confirmed in each mouse by the increase of Pc in comparison to the control of PBS administration. The results are shown as the mean with or without 1 ϫ SE. The percentage of RAG1/GFP positive cells is shown after gating the peritoneal B-1 and B-2 cells as described in Materials and Methods. b Number of cells recovered from peritoneal cavity ϫ106.

information that is in contrast with previous reports of rag1 gene 8. Yagi, T., S. Nada, N. Watanabe, H. Tamemoto, N. Kohmura, Y. Ikawa, and up-regulation in Pc B-1 cells (7). The postulated association of S. Aizawa. 1993. A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene. Anal. Biochem. 214:77. Downloaded from RAG reexpression with the tendency of autoreactivity of the 9. Han, S., B. Zheng, D. G. Schatz, E. Spanopoulou, and G. Kelsoe. 1996. Neoteny B-1 repertoire have now need to be re-evaluated at least in the Pc in lymphocytes: Rag1 and Rag2 expression in germinal center B cells. Science B-1 cells. 274:2094. 10. Schlissel, M., A. Constantinescu, T. Morrow, M. Baxter, and A. Peng. 1993. Double-strand signal sequence breaks in V(D)J recombination are blunt, 5Ј-phos- Acknowledgments phorylated, RAG-dependent, and cell cycle regulated. Genes Dev. 7:2520. 11. Meffre, E., F. Papavasiliou, P. Cohen, O. de Bouteiller, D. Bell, H. Karasuyama,

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