receptor editing

8/14/2019 Receptor Editing

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Receptor Editing in Peripheral B Cell ToleranceAuthor(s): Jeffrey S. Rice, Jeffrey Newman, Chuansheng Wang, Daniel J. Michael, BettyDiamond and Matthew D. ScharffSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 102, No. 5 (Feb. 1, 2005), pp. 1608-1613Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/3374478 .

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eceptor e i t i n g n per iphera l el l toleranceJeffrey S. Rice*t, Jeffrey Newman*t, Chuansheng Wang*, Daniel J. Michael*, and Betty Diamond*t?

Departments of *Microbiology and Immunology and tMedicine, Albert Einstein College of Medicine, Bronx, NY10461

Communicated by Matthew D. Scharff, Albert Einstein College of Medicine, Bronx, NY, December 13, 2004 (received for review November 10, 2004)

Receptor editing or secondary Ig gene rearrangement occurs inimmature, autoreactive B cells to maintain self-tolerance. Here weshow that nonspontaneously autoimmune mice immunized with apeptide mimetope of DNA develop peptide- and DNA-reactiveantibodies. Antigen-specific B cells display a follicular B cell phe-notype. As these cells move into the memory compartment, manyexpress RAG protein and acquire expression of both c and Alightchains. Thus, this study provides evidence for receptor editingoccurring n a mature, antigen-activated Bcell population. Becausethe receptor editing observed here occurred in an autoreactiveresponse to antigen, it may function o maintain peripheral olerance.

autoimmunity I B cell selection I autoantibodies

utoreactive B cells routinely arise during the immuneresponse to foreign antigen. Although it has been demon-

strated that the processes of apoptosis, anergy, and receptor

editing maintain tolerance in immature B cells, it is clear thatautoreactivity can also arise in mature B cells in a germinalcenter response (1-4). Mechanisms that limit autoreactivity inthis population are less well characterized. Studies investigatingtolerance induction of antigen-specific B cells within a nativerepertoire have been limited by the rarity the population. Inmurine models of lupus, DNA-reactive B cells represent


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Fig. 1. Differentiation and maturity of tetramer-reactive plenocytes. (A-C)Representative etramer staining after secondary challenge in the spleen (A)andinhibition of splenic staining by 30 mM nonfluorescent etramer (B)and bone marrow C). D) Histogram f intracellular gG taining in tetramer populations.Thick line, tetramer?B22010w; hin line, tetramer+B220hi; otted line, tetramer-B220hi; haded line, tetramer-B220-. (E) Expression of AID in tetramerpopulations as determined by real-time PCR. F)Expression f Bcl-6 n tetramer populations as determined by real-time PCR.Data in A-D are representative of10 mice. Real-time PCR n E and F is representative of three independent experiments.

Sorting was performed on a MoFlo (DakoCytomation, FortCollins, CO).

Histology. Spleens were removed on day 15 and frozen in

Tissue-Tek OCT Compound (Miles, Elkhart, IN). Staining wasperformed in 3% FCS + 0.2% Triton X-100 for 30 min at roomtemperature. Tetramer staining was performed at 4?C overnight.Slides were mounted in Aqua-Poly/Mount (Polysciences, Inc,Warrington, PA). Fluorescent microscopy was performed on anAxioCam II (Carl Zeiss Microimaging, Thornwood, NY) withAxiovision 3.1. The following antibodies were used: AlexaFluor488-anti-GFP (rabbit polyclonal, Molecular Probes), bi-otin-anti-CD11b (clone M1/70, Pharmingen), PE-anti-B220(RA3-6B2, Pharmingen), anti-RAG1 (G109-256, Pharmin-gen), anti-RAG2 (rabbit polyclonal, Pharmingen), streptavidin-AMCA (Vector Laboratories). Anti-RAG antibodies were la-beled by using Zenon Alexa Fluor 350 anti-mouse IgG2b oranti-rabbit IgG, and Alexa Fluor 488 anti-rabbit IgG (MolecularProbes).

Adoptive Transfers. For RAG2:GFP+/- transfers (see Fig. 3E),spleens were removed from naive donor mice and lymphocytesisolated as described above. Mice received 4 x 107 cells each byretro-orbital injection. Two weeks after transfer, recipient micewere immunized according to the protocol described. For fur-ther details, see Supporting Text.

Real-Time PCR. RNA was isolated from sorted cells by usingTriZol reagent (Invitrogen) following the manufacturer's rec-ommendations. Random hexamer-primed RT-PCR was per-formed on 5 Al of RNA by using SuperScript II reverse tran-

scriptase (Invitrogen) in 100-pJ inal volume, according to themanufacturer's protocol. Real-time PCR was performed byusing an ABI 7900 (Applied Biosystems) and analyzed by usingSDS version 2.2. ABI Gene Expression Assays were used, and the

reactions were performed by using TaqMan Universal PCRMaster Mix in 10-pA inal volume. Standard curves were made foreach experiment by using total spleen and bone marrow cDNA.Relative template concentration was determined from the stan-dard curve by using Cts determined by the SDS software. Allprimers used spanned an intron/exon border. ABI Primer Idswere: RAG2, MmO0501300_ml; AID, MmOO50774_ml; Bcl6,MmO0477633_ml; RNA pol2a, MmO0839493ml.

ResultsPhenotypic Analysis of the Tetramer-Reactive B Cells. After immu-nization with DWEYS-MAP, tetramer-reactive splenic B cellscan be identified with fluorescently tagged antigen (15). Twopopulations are observed: one population with high B220 ex-pression (B220hitetramer+), and a second population, compris-

ing the majority of tetramer-reactive cells, with low B220 ex-pression (B220lOwtetramer+) (Fig. 1A). Extensive studies oftetramer-reactive cells demonstrate that tetramer binding occursonly in mice immunized with the DWEYS-peptide, and isinhibitable by unlabeled tetramer (Fig. 1B). We have previouslyconfirmed their B cell lineage (15). Interestingly, theB220lOwtetramer+ opulation is expanded in the bone marrow(Fig. 1C). Intracellular expression of IgG was observed in bothtetramer-reactive populations (Fig. ID). GL7 was highly inducedon the B220hi population (Table 1, which is published assupporting information on the PNAS web site), and real-timePCR analysis detected AID expression in this population (Fig.

Rice et al. PNAS I February 1, 2005 | vol. 102 | no. 5 | 1609

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Fig. 2. RAG expression in tetramer-reactive B cells. (A and B) Representative histogram of GFP expression on day 15 in B220+HSAIOw DWEYS+ (A) and 1O-2+(B) B cells from RAG2:GFP Tg mice (solid line) compared to B220+HSAlOwtetramer+ B cells in wild-type mice (shaded line). GFP expression is not seen inB220+HSAlOwtetramer- B cells in RAG2:GFP Tg mice (Insets). (C) Time course of GFP expression in DWEYS-immunized RAG2:GFP Tg mice versus wild-type mice.

1E). Finally, Bcl-6 was found to be expressed in both the B220Oand B220'Owtetramer+ opulations (Fig. iF). These findings areconsistent with a germinal center (16-19) and a memory B cellpopulation (20). Additional phenotypic analysis (Table 1) sup-ports this interpretation, and suggests that the B220hi populationcontains precursors of the B22010w opulation.

RAGExpression s Induced n Antigen-Binding Cells.To determinewhether there was any attempt to regulate the autoreactivityarising in response to DWEYS-MAP immunization, we lookedfor evidence of receptor editing in tetramer-reactive B cells. Weused mice with a functional RAG2/GFP fusion gene insertedinto the endogenous RAG2 locus (21) that allows for tracking ofRAG2-expressing cells by their GFP fluorescence while main-taining the degradation sequences present on the wild-typeprotein and critical to ensuring a normal pattern of RAGexpression. BALB/c x RAG2:GFP Fl mice were immunizedtwice with DWEYS-MAP or an irrelevant peptide, 10-2-BSA,and mature splenic B cells (B220hi HSAlOw)were analyzed forGFP expression. By day 15 after immunization, DWEYS-specificB cells expressed GFP (Fig. 2A), but the 10-2-specific population

did not (Fig. 2B). No GFP expression was observed in mature Bcells that were not activated by antigen (B220hi HSAlOwtet-ramer-, Fig. 2A and B Insets). Kinetic studies revealed that GFPexpression arose >12 days after immunization, and declined by22 days (Fig. 2C). Thus, GFP expression occurred only in matureB cells specific to the dsDNA mimetope, was absent in matureB cells specific to a non-self peptide (Fig. 3A), and displayed akinetics consistent with antigen induction. To confirm RAG2expression, splenocytes were transferred from RAG2:GFP miceinto SCID recipients. Recipient mice were rested for 2 weeks toallow for engraftment. Because there is no emigration of new B

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Fig. 3. RAG expression in autoreactive, mature B cells. (A) GFP expression inB220+HSA'Owtetramer+ B cells from DWEYS filled circles) and 10-2 (open circles)immunized RAG2:GFP Tg mice. (B) GFP expression in B220+HSAl?wtetramer+ Bcells after adoptive transfer and challenge.

cells from the bone marrow n SCID mice, the B cell populationin the recipient mice consists exclusively of mature cells (22).Mice were immunized and GFP expression was observed n thetetramer+ population, confirming that the expression of theRAG2:GFP usion protein can occur n mature B cells (Fig. 3B).

Additional evidence for antigen-induced RAG2 expression

was acquired by histological analysis f the spleen. GFP-positivecells were present primarily n the red pulp of DWEYS-MAPimmunized mice (Fig. 4C) but not in naive (Fig. 4A) or 10-2-immunized (Fig. 4B) littermates. Staining of DWEYS-MAPimmunized BALB/c mice with anti-RAG1 (Fig. 4D) and anti-RAG2 (Fig. 4E) antibodies demonstrated hat both proteinswere coexpressed in splenic B cells of DWEYS-MAP-

Fig. 4. Histological analysis of RAG2:GFP and BALB/c mice. (A-C)RAG2:GFP+I- pleen sections stained with anti-B220 (red), anti-CD1 lb (blue),and anti-GFP (green) in naive (A), 10-2-immunized (B), and DWEYS-immunized (C) mice. Images were obtained by using the x 10 objective. (D-F)BALB/c spleen sections from DWEYS-immunized mice stained with anti-RAG1(D) oranti-RAG2 (E), and superimposed (F). Images were obtained by using thex40 objective.

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Fig. 5. Histological staining with fluorescent tetramers. (A and B) BALB/cspleen sections from 10-2 (A) or DWEYS B) immunized mice stained withanti-B220 red) and DWEYS-A488 green). Images were obtained by using thex10 objective. (C-F) BALB/c pleen sections from DWEYS mmunized micestained with anti-B220 (C,red), DWEYS-A488D, green), anti-RAG2 E, blue),and superimposed (F). Images were obtained by using the x40 objective.

immunized mice (Fig. 4F), but not in naive or 10-2-immunizedBALB/c or RAG2:GFP+/- mice (data not shown).

Staining by the DWEYS tetramer was observed again in thered pulp of BALB/c mice immunized with DWEYS-MAP Fig.SA) and not in mice immunized with 10-2 (Fig. SB) or in naivelittermates (data not shown). Some DWEYS-reactive B cells

were present n B cell follicles; hese cells were IgM+ (data notshown). Costaining with anti-B220 Fig. 5C), DWEYS-tetramer(Fig. SD), and anti-RAG2 (Fig. 5E) revealed that RAG2 ex-pression was limited to DWEYS-reactive B2201 cells (Fig. SF).Consistent with the flow cytometry data, no RAG expressionwas observed in cells not binding to the tetramer.

Real-Time PCR Analysis of RAG2 Expression. To confirm RAG2expression, mature B cells (B220?AA4.11OWHSAIOw) were iso-lated from DWEYS-MAP or 10-2-BSA mmunized and naiveBALB/c mice. Real-time PCR for RAG2 revealed expression fRAG2 RNA only in splenocytes solated from DWEYS-MAPimmunized mice (Fig. 6A), and was specific to mature B cells(Fig. 6B).

Receptor Editingn Tetramer-Reactive ells.

Receptor editingleaves

characteristic molecular ignatures, ne of which s coexpressionof K and A light chains (23). Therefore, we investigated hepattern of light chain expression on tetramer-reactive cells.Fig. 7A shows that a majority 81.3%) of the tetramer-reactivecells coexpressed K and A ight chains. A ight chain coexpressionwas related to antigen-induced ifferentiation because


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Fig. 7. Analysis of light chain expression. (A) Bivariate plots depicting A and K staining of DWEYS etramer-gated cells. (B)Bivariate plots depicting K and Astaining of 10-2 tetramer-gated cells. (C)Bivariate plots depicting Aand K staining of DWEYS etramer gated cells from SCID ecipients of BALB/c plenocytes.

one that was B220hi and the other B22010w. he B220hitetramer?population demonstrated high levels of costimulatory molecules,consistent with recent activation, whereas the B220lOwtetramer?population expressed lower levels of these markers.B220hitetramer? cells also express germinal center markers.Phenotypic characteristics of the populations suggest movementfrom the B220hi to the B22010w ompartment. Although it hasbeen suggested that the B22010w ntigen-specific cells can resultfrom antibody bound by Fc receptors on non-B cells (25), wepresent evidence that the antigen-specific cells in our model areB cells (Figs. 9 and 10, which are published as supportinginformation on the PNAS web site). We show a lack of tetramerstaining B cells despite high serum antibody titers in naive micegiven immune serum, and tetramer staining B cells despite a lackof serum antibody titers in naive mice given immune B cells byadoptive transfer.

The B220lOwtetramer? opulation possesses many character-istics of a memory B cell population. McHeyzer-Williams andcolleagues (26, 27) reported a B2201owmemory population in

NP-immunized mice. A B22010wmemory population has beenobserved also in the quasimonoclonal mouse (28), and humanmemory B cells have been shown to down-regulate B220 ex-pression (29, 30). Furthermore, it has been suggested that Bcl-6,which is expressed in the B22010wpopulation,. is involved inmemory B cell maintenance (20). The data in Table 1 furthersuggest that the B220lOwtetramer+ population contains twosubsets of B cells: one that differentiates into plasma cells, andone that becomes memory cells. In accordance with this, a subsetof the B220lOwtetramer? ells express CD138 (data not shown)and is present in the bone marrow.

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Fig. 8. Expression of activated caspase-3 and A light chain. (A) Activecaspase-3 expression in B220+ (circles), DWEYS+A- (squares), and DWEYS+A+B cells (triangles). (B) Densitometric quantitation of A ight chain/actin RNA asdetermined by semiquantitative RT-PCR n the indicated sorted populations.

Secondary rearrangement of antibody genes in immature Bcells in the bone marrow is well established. Evidence forsecondary rearrangements in mature B cells has been contro-versial. Studies of murine germinal center B cells have shownexpression of RAG protein by immunohistology (31) and RAGmRNA by RT-PCR and Southern blotting (32, 33). Activation ofsplenic B cells by IL-4 and LPS or CD40L has also been shownto induce RAG expression (33). Studies of healthy humansubjects have found evidence of RAG expression in germinalcenter centrocytes and memory B cells (23, 34, 35). RAGexpression has also been found in B cells in the synovial tissueof rheumatoid arthritis patients and in the peripheral blood Bcells of SLE patients (36, 37).

Recently, three lines of RAG-indicator mice have been de-veloped (21, 38, 39). Each line permits monitoring of RAGexpression (either RAGI or RAG2) by linking RAG expressionto GFP expression. In each of the GFP-RAG indicator models,the authors identify peripheral B cells expressing RAG, butconclusive assignment to the mature compartment was notpossible. The failure to demonstrate a mature population of Bcells undergoing receptor editing is likely to be a reflection of therarity of such cells. In the study by Monroe et al. (21), GFPexpressing antigen-specific B cells with markers of germinalcenter cells were seen when an antigen-specific population wasanalyzed. Although it was postulated that these may be immatureB cells recruited to the germinal center response, the data werealso consistent with reexpression of RAG genes in mature Bcells.

To confirm in our study that receptor editing was occurringselectively in B cells responding to antigen, we immunizedRAG2:GFP x BALB/c F1 mice with DWEYS-MAP. Thesemice express a GFP-RAG2 fusion protein under the direction ofthe RAG2 promoter, as described by Monroe et al. (21). Maturetetramer-binding B cells expressed GFP, whereas the mature Bcells that did not respond to peptide showed no expression ofGFP. Thus, RAG expression is linked to antigen activation. GFPexpression was present for a short time after antigenic challengein a model of induced autoreactivity, but not in a nonautoreac-tive response, suggesting that receptor editing is a mechanism togenerate peripheral tolerance and that there is a limited periodduring which mature antigen-activated B cells can undergo thisprocess. Moreover, the response to the dsDNA mimetope andthe induction of RAG correlated with the coexpression of K andAby tetramer-binding cells. We confirmed by adoptive transferthat RAG expression occurred in mature cells in this system. Wespeculate that B cells leaving the germinal center can, liketransitional B cells, be induced to undergo receptor editing and

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apoptosis if they encounter soluble antigen. This might accountfor the occurrence of receptor editing in autoreactive, but notnonautoreactive, cells.

In summary, the data presented here demonstrate the impor-tance of studying the emergence of autoreactivity in wild-typeanimals in an antigen-specific manner. The ability to examinerare populations of cells that have a particular antigenic speci-ficity will allow us to probe B cell regulation to a degree notpreviously possible. We can now recognize that as follicular Bcells differentiate in an antigen-induced response, negative

selection of autoreactive cells occurs concomitant with thetransition to the memory or plasma cell compartment. Becauseour initial observation that somatic mutation can lead to thegeneration of autospecificity in B cells (1), we and others haveshown that autoreactive B cells are generated at high frequencyduring the response to certain antigens (2, 3). Previous studieshave also demonstrated that desnite the activation of autoreac-

tive B cells in a primary response to antigen, there is an absenceof these cells in the memory B cell population (40, 41). Themethodology used in the current study reveals that receptorediting is responsible for the elimination of autoreactivity thatoccurs in antigen-activated B cells. RAG expression and editingmay lead to translocations of the Ig locus that contribute to B cellmalignancy in germinal center cells, and may explain why suchtumors are often autoreactive.

This study demonstrates that the processes that govern neg-ative selection after diversification of the repertoire in naive Bcells may also operate in the antigen activated B cell repertoire.The ability of mature B cells to undergo secondary Ig rearrange-ment confirms that the mechanisms important in the mainte-nance of tolerance in immature B cells also function in themature population.

We thank Kristie Gordon n the Fluorescence Activated Cell SortingFacility at Albert Einstein College of Medicine.

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