Dominant, Hierarchical Induction of Peripheral Tolerance ... - Cell Press

Immunity, Vol. 17, 317–327, September, 2002, Copyright 2002 by Cell Press

Dominant, Hierarchical Induction of Peripheral Tolerance during Foreign Antigen-Driven B Cell Development Evangelia Notidis,2 Lynn Heltemes,2 and Tim Manser1 Kimmel Cancer Center Thomas Jefferson University Philadelphia, Pennsylvania 19107

Summary We created mice expressing transgene-encoded BCRs with “dual reactivity” for the hapten Ars and nuclear autoantigens. Expression of transgene-encoded BCRs was not evident in the memory compartment despite observation of transgene-expressing B cells in germinal centers following Ars immunization. In contrast, dual reactive mAbs were readily obtained from mice with enforced expression of Bcl-2 following secondary Ars immunization. However, while these mAbs were hypermutated and displayed increased affinity for Ars, all had reduced avidity for DNA and intracellular autoantigens. Thus, Bcl-2 alters dominantnegative selection of dual reactive B cells during the Ars response, but this is restricted to those with lowered autoreactivity, demonstrating a hierarchy of peripheral tolerance during memory B cell development. Introduction Developing B cells with antigen receptors (BCRs) that bind self-antigens in the bone marrow may be deleted by apoptosis (Chen et al., 1995; Nemazee and Burki, 1989) or undergo receptor editing (Gay et al., 1993; Tiegs et al., 1993). However, not all autoreactive B cells are eliminated by these processes, as the primary peripheral B cell compartment in nonautoimmune mice is characterized by the expression of multireactive BCRs that bind a variety of self-antigens (Limpanasithikul et al., 1995; Rousseau et al., 1989) and of autoreactive B cells with specificities for autoantigens not expressed in the bone marrow (Litzenburger et al., 1998; Murakami et al., 1992). In addition, considering the rate of variable (V) region mutation in the germinal center (GC), B cells expressing autoreactive antibodies must frequently arise during foreign antigen-driven immune responses (Casson and Manser, 1995; Diamond et al., 1992). Autoreactive peripheral B cells are often argued to be ignorant of self-antigens due to low concentrations or sequestration of their cognate autoantigens (Goodnow, 1992; Nossal, 1993). However, it has become evident that tolerance to peripheral autoantigens can be actively imposed, resulting in ultimate cell death (Monroe, 1998; Rathmell et al., 1995), anergy (Erikson et al., 1991; Goodnow et al., 1988), or follicular exclusion (Cyster et al., 1994; Mandik-Nayak et al., 1997). The developmental potential of autoreactive peripheral B cells subjected to such active regulation remains controversial. Some view 1 2

Correspondence: [email protected] These authors contributed equally to this work.

peripheral B cell tolerance as irreversible, resulting in a subpopulation of B cells which, although physically present, is functionally incompetent (Cooke et al., 1994; Nguyen et al., 1997). However, recent studies have demonstrated that provision of strong BCR signals and either genuine or surrogate T cell help can result in the activation of self-antigen-regulated B cells (Noorchashm et al., 1999; Sater et al., 1998; Seo et al., 2002). Thus, selftolerant B cells with BCRs that are “dual reactive” for both self and foreign antigen could theoretically be recruited to participate in a foreign antigen-driven immune response. This may occur when a response is driven by a foreign antigen that contains a B cell epitope to which an autoreactive B cell is crossreactive. Indeed, the etiologies of certain autoimmune diseases have been suggested to be a consequence of foreign antigen mimicking self-antigen (Oldstone, 1998). In this regard, many pathogens express epitopes that are immunologically crossreactive with self-epitopes (reviewed in Karlsen and Dyrberg, 1998). Also, a foreign antigen that is bound to a self-antigen could stimulate peripheral autoreactive B cells via recruitment of T cell help (Krishnan and Marion, 1993; Moens et al., 1995; Parker and Eynon, 1991). For these reasons, it has been argued that peripheral tolerance mechanisms must exist that are operative during the B cell response (Hande et al., 1998; Linton et al., 1991). However, whether self-antigen-regulated B cells are prevented from contributing to the memory compartment due to such tolerance mechanisms has not been adequately examined. We have shown that B cells with low-affinity, multireactive antigen receptors can participate in a foreign antigen-driven immune response (Hande et al., 1998). The antibodies that dominate the late primary and anamnestic responses to the hapten p-azophenylarsonate (Ars) in A strain mice are encoded by a single combination of V gene segments (termed canonical). Prior to somatic mutation, they differ only at VL-JL and VH-D-JH junctions and exhibit weak binding to nuclear and cytoplasmic structures, as well as DNA (Manser et al., 1987; Naparstek et al., 1986). In contrast, hypermutated canonical antibodies expressed by memory B cells have increased affinity for Ars but do not detectably bind self-antigens (Naparstek et al., 1986). These data suggest that both positive and negative selection, acting in conjunction with V region hypermutation, purge the responding B cell population of autoreactivity. Consequently, we developed the specificity maturation hypothesis which contends that the driving force behind the maturation of the B cell response is specificity for the foreign antigen rather than affinity alone (Casson and Manser, 1995; Hande et al., 1998). However, due to the low affinity of unmutated, canonical V regions for self-antigens, we considered that B cells expressing such V regions were functionally ignorant of these antigens prior to entering the memory B cell pathway. Therefore, we sought to create a model system in which B cells expressing canonical V regions with strong self-reactivity were members of the peripheral compartment. To this end, we exploited our previous finding that

Immunity 318

Figure 1. Autoreactivities Conferred to Canonical V Regions by the R55 Mutation (A) The R55 mutation confers ssDNA reactivity to canonical V regions that is comparable to that displayed by PL9-6, while the dsDNA reactivity is ⵑ4-fold lower than PL9-6. The R55 mutation also confers chromatin reactivity, which is completely lacking in the unmutated canonical mAb, 36-65/223. (B) Injecting transfectoma cells secreting the R55/223 mAb into pristane-primed SCID mice results in immune complex deposition in kidney glomeruli, but injection of 36-65/223 transfectoma cells does not. Original magnification of images was 100⫻. (C) The R55 mutation confers nuclear, perinuclear, and cytoplasmic reactivities to canonical V regions as determined by ANA assays. Note the strong binding to mitotic figures. A similar analysis using the unmutated canonical mAb 36-65/223 gave negative results even at 25 ␮g/ ml of purified mAb. Original magnification of all images was 250⫻.

an arginine (R) mutation at position 55 in VHCDR2 of canonical V regions substantially increases binding to DNA without affecting affinity for Ars (Hande and Manser, 1998) and generated transgenic mouse lines using a canonical VH region gene containing the R55 mutation. This allowed us to address whether truly dual reactive B cells could be recruited into the immune response and, if so, whether they could contribute to the memory B cell compartment via specificity maturation. Results The R55 Mutation Confers Chromatin, ANA, and Increased DNA Reactivity An Ig expression vector encoding a canonical H chain with the R55 mutation (R55/223) was transfected into the hybridoma line 36-65L (Sharon et al., 1984) expressing a canonical light (L) chain but no heavy (H) chain, resulting in the production of a canonical mAb with only this

mutation. The reactivity of this mAb was examined by ELISAs, as well as ANA assays (Figures 1A and 1C) and Ars-tyrosine fluorescence quenching (see below). The R55 mutation confers a substantial increase in reactivity for DNA. The ssDNA reactivity was comparable to that of PL9-6, an anti-chromatin antibody obtained from a diseased MRL mouse (Losman et al., 1993) while dsDNA binding was approximately 4-fold lower than PL9-6 (Figure 1A). More strikingly, the R55 mutation conferred chromatin binding and other intracellular autoantigen reactivities, which are not exhibited by unmutated IgG canonical antibodies (Figure 1). The R55/223 mAb stained nuclear, perinuclear, and cytoplasmic structures (Figure 1C). Furthermore, injecting the R55/223 transfectoma into SCID mice resulted in immune complex deposition in kidney glomeruli. There was no evidence of such deposition in SCID mice injected with the 36-65/ 233 (unmutated canonical) transfectoma (Figure 1B). Finally, the R55 mutation did not significantly alter affinity for Ars (see below).

Memory B Cell Tolerance 319

Figure 2. Somatic and Germline Modification of the Endogenous IgH Locus by R55VH Transgenes (A) The VR55 and VER55 transgene structure and schematic representation of the nonreciprocal recombination event leading to the generation of transgene-hybrid H chain loci is shown. This recombination occurs in B cells as the result of homologous pairing between a region 3⬘ of the coding VDJ of one transgene copy in the transgenic array and the analogous region in the IgH locus. The homologous pairing and recombination site is indicated with solid crossed lines, and the nonhomologous recombination site is indicated with dotted crossed lines. The result of this recombination event is the integration of the transgenic VH region into the endogenous IgH locus. (B) The structure of the HKIR R55 targeting vector and the recombination events leading to the generation of the R55VH germline knockin IgH locus are illustrated. The introduction of novel StuI sites into the IgH locus via the incorporation of R55 VH DNA allows a Southern blot assay for this locus to be performed using the J14C-S probe (location shown with a black bar).

Generation and Characteristics of Transgenic Mice To create mice expressing canonical BCRs with the R55 mutation, two approaches were taken. Lines of transgenic mice, termed VR55 and VER55, were generated using a canonical R55VH region gene. The Ig transgenes used did not contain constant region gene(s) and therefore could not encode complete H chains. Our previous studies have shown that VH transgenes of this type integrate into the germline IgH locus via a somatic interchromosomal one-sided homologous recombination event, illustrated in Figure 2A (Giusti et al., 1992). The formation of the resulting hybrid H chain loci takes place at a frequency comparable to individual endogenous VH gene rearrangements, resulting in only a subtle alter-

ation of the B cell repertoire (Giusti et al., 1992). Transgene hybrid H chain loci can undergo somatic hypermutation and isotype class switching (Giusti et al., 1992). The above transgenic mice allowed analysis of the behavior of canonical R55 VH-expressing clonotypes in a nearly normal physiological context, where B cells expressing canonical BCRs encoded by endogenous genes also participate in the anti-Ars response (Giusti et al., 1992). However, the extremely low precursor frequency of transgene-expressing canonical clonotypes in such mice precludes quantitative analysis of their location and phenotype prior to and after primary immunization (Vora et al., 1999). Therefore, more conventional lines of canonical R55VH IgH locus knockin transgenic

Immunity 320

Table 1. Summary of B Cell Fusions from VR55 and VER55 Splenocytes # Fusions

# Fusions

Founders

Primary

Secondary

Tertiary

# E4⫹ Hybridomas

VR55-50 VR55-46 VR55-32 VR55-54 VER55-89 VER55-102 VER55-148 VER55-142

3 5 5 4 –e – – –

3 – 6 – – 4 – 2

– – 5 – – – – –

11 2 13 0 – 30 – 2

0 0 0 0 – 0 – 0

9 5 4 3 11 1 8 –

9 8 5 13 27 3 38 –

Totals

17

15

5

58

0

41

105

1

BVER55

–

4

–

34

13

–

–

–

a

b

b

# Transgene⫹ Hybridomasd

Hyperimmunizationc

# E4⫹ Hybridomas

# Transgene⫹ Hybridomas 0 0 0 0 1 0 0 –

Primary fusions were performed at days 4, 7, 10, 12, and 15 postimmunization with 100 ␮g of Ars-KLH emulsified in CFA. For secondary immune responses mice were boosted with 50 ␮g of Ars-KLH in PBS at least 1 month after priming, while for tertiary immune response mice were boosted with 50 ␮g of Ars-KLH in PBS at least 1 month after priming and then once again 1 month later. All B cell fusions were performed 3 days following the final boost. c Mice were primed with 100 ␮g of Ars-KLH emulsified in CFA, boosted 1 week later with 50 ␮g of Ars-KLH in PBS, and then boosted with 50 ␮g of Ars-KLH in PBS three more times every other day. Fusions were performed 3 days following the final boost. d All of the B cell hybridomas generated were screened for reactivity with the anti-idiotypic antibodies E4 and 107; transgene expression status was confirmed by RT-PCR sequence analysis. e Dashes (–) indicate no fusions performed. a

b

mice, termed HKIR mice, were generated via homologous recombination in ES cells. Figure 2B illustrates the approach taken and the structure of the resulting knockin germline IgH locus (see Experimental Procedures for details). Transgenic R55VH Regions Are Rarely Expressed by Canonical Hybridomas Derived from ArsImmunized VER55 and VR55 Mice Splenic B cell hybridomas were generated from VR55 and VER55 mice at various times after primary, secondary, tertiary, and hyperimmunization with Ars-KLH. Their expression of canonical mAbs was evaluated using the monoclonal anti-idiotypic Ab E4, which recognizes most canonical VH regions (Hiernaux et al., 1983; Manser and Gefter, 1986), and the monoclonal anti-idiotypic Ab 107, highly specific for the transgenic VH region (Giusti et al., 1992; Weissman et al., 1985). Based on the yield of E4⫹ hybridomas, there was a normal frequency of canonical B cell participation in the anti-Ars response (Table 1). However, only one of the 163 canonical splenic hybridomas generated from a total of 78 VR55 and VER55 mice expressed the transgenic VH region (Table 1). The mAb expressed by this hybridoma bound Ars with avidity comparable to 36-65, the prototypical canonical mAb (Marshak-Rothstein et al., 1980), but did not bind ssDNA or dsDNA as determined by ELISA. Sequence analysis of the transgenic VHR55 gene expressed by this hybridoma showed that the R55 codon was unaltered. However, there were secondary mutations in the VH region that must account for the attenuation of DNA binding displayed by this mAb (data not shown). Consistent with the extremely low yield of hybridomas from VR55 and VER55 mice that expressed the transgenic R55VH, 107⫹ serum antibody was low or absent in VR55 mice following Ars immunization (data not shown). Two of five VR55 mice analyzed displayed a slight increase in 107⫹ serum antibody during secondary and

tertiary immune responses, but the levels were substantially lower than those previously observed (Giusti et al., 1992) in other animals transgenic for the unmutated canonical VH region. The extremely infrequent isolation of hybridomas expressing canonical R55VH antibodies from Ars-immunized VR55 and VER55 mice could have resulted from the inability of their B cell precursors to escape a tolerance checkpoint prior to the Ars response. To address this issue, we analyzed primary development of canonical R55VH B cells in the HKIR mice. Development in the bone marrow appeared normal as compared to littermate controls (data not shown). As shown in Figure 3A, most if not all HKIR E4⫹ splenic B cells do not express an endogenous ␮ chain. Moreover, a substantial fraction of HKIR spleen cells stained with the E4 mAb (data not shown), demonstrating that B cells expressing canonical R55VH BCRs can exit the bone marrow and populate the periphery. In addition, such B cells exhibit what appears to be a follicular phenotype, as defined by CD21, CD23, and CD24 expression (Figures 3B and 3C). These results were corroborated via immunohistochemical analysis, revealing numerous E4⫹ cells throughout follicular areas (data not shown). Nonetheless, flow cytometric analyses showed that both sIgM and sIgD levels were substantially reduced on E4⫹ HKIR splenic B cells relative to the majority of B cells in these mice (Figure 3D). Next, we performed immunohistochemical analysis of the spleens of VER55 mice at various times after primary immunization with Ars-KLH. As expected from our previous studies of A/J mice (Vora et al., 1999), E4⫹ cells were infrequently detected in the spleens of these mice, and then only in GCs. As illustrated in Figure 4A, some of these GCs contained multiple E4⫹ cells that also stained with the 107 mAb, indicating the presence of canonical R55VH transgene-expressing B cells. To increase the frequency of canonical R55VH-expressing B cells partici-


Figure 3. Most Canonical R55VH B Cells Display a Follicular Phenotype but Express Reduced Surface Levels of BCR HKIR mice were crossed to C57BL/6 mice, and offspring containing (HKIR) and lacking (littermates) the targeted allele were identified. Splenocytes from both were evaluated for expression of surface markers via flow cytometry. (A) B220⫹ or E4⫹ cells were assayed for expression of the IgM allotype encoded by the knockin allele (IgMa) or the endogenous C57BL/6 IgH locus (IgMb). (B) B220⫹ or E4⫹ cells were evaluated for expression of CD21/35 and CD23 to allow distinction of marginal zone (MZ) versus follicular B cells. Equivalent gates were set around the MZ (upper left) and follicular (lower right) B cell subpopulations. Note that E4⫹ HKIR B cells are not enriched in the MZ subpopulation (CD21high,CD23low). (C) B220⫹ and E4⫹ cells were evaluated for levels of expression of CD24 (HSA) and CD21/ 35 to allow quantitation of T1 and T2 transitional B cell subcompartments. Equivalent gates were set around these (lower right, T1; upper right, T2) and the follicular B cell subpopulation (left). (D) B220⫹, E4⫹, or total cells in the lymphocyte gate were analyzed for surface levels of IgM and IgD. Note that while the bulk lymphocyte population in HKIR mice displays a fairly normal IgM versus IgD profile as compared to littermates, the majority of E4⫹ cells in these mice display substantially reduced levels of both surface IgM and IgD.

pating in the response, we transferred HKIR splenocytes to syngeneic recipients and immunized them with ArsKLH. Twelve days later, vigorous GC and AFC responses were apparent in the spleens of the recipient mice, and E4⫹ cells could be observed in many GCs (Figure 4B). Enforced Expression of Bcl-2 Results in a Dramatic Increase in Splenic Hybridomas Expressing the Transgenic R55VH Region Enforced expression of Bcl-2 in the B cell compartment of A strain mice from a transgene results in a large decrease in the level of apoptosis in the GC and in the ability of B cells expressing canonical BCRs with increased affinity for both Ars and DNA to enter the Ars-induced memory B cell compartment (Hande et al., 1998). To test whether Bcl-2 could alter the negative selection of B cells expressing the transgenic R55VH

region, VER55 mice were crossed to A.Bcl-2 mice (Hande et al., 1998) resulting in a double transgenic line called BVER55. Transgene-expressing splenic B cell hybridomas were readily obtained from BVER55 mice. Nearly one-third of the secondary hybridomas from ArsKLH-immunized BVER55 mice expressed the transgenic R55VH region (Table 1). Furthermore, approximately 50% of the hybridomas generated from three out of four BVER55 mice expressed the transgene encoded R55VH region. The R55 Mutation in Canonical VH Regions Does Not Affect Somatic Hypermutation or Affinity Maturation to Ars in Bcl-2 Transgenic Mice We previously concluded that enforced expression of Bcl-2 did not alter somatic hypermutation or affinity maturation of endogenous canonical V regions (Notidis et

Immunity 322

Figure 4. Canonical R55VH B Cells Are Evident in GCs during a Primary Ars Response (A) Parallel spleen sections from a VER55 mouse at day 14 postimmunization with Ars-KLH were stained with 107, which recognizes the transgenic VHR55 region, and either GK1.5, which delineates CD4⫹ T cells, or PNA, to elaborate GCs. Another parallel section was also stained with E4 to delineate canonical B cells, and also with PNA. An analysis of 11 VER55 mice sacrificed at various times after Ars-KLH immunization revealed that out of 19 E4⫹ GCs, 5 were also 107⫹. Original magnification of images was 100⫻. (B) Splenocytes from HKIR mice were transferred to syngeneic recipients, and the recipients were immunized with Ars-KLH and sacrificed 12 days thereafter. Spleens were taken, processed for histology, and stained as described in the Experimental Procedures. Several examples of GCs containing numerous E4⫹ cells are illustrated. The results are representative of those obtained from four chimeric mice. Original magnification of images was either 100⫻ or 250⫻. (C) Littermate spleen sections were stained with either 107 or E4 and GK1.5 under the same conditions as used above to evaluate background levels of staining with these mAbs. For unknown reasons, central arterioles stain blue with developing reagent. Original magnification was 100⫻.

al., 2001). To evaluate whether the R55 mutation affected these processes, we conducted sequence analysis of the VH and V␬ genes expressed by the canonical hybridomas from BVER55 mice. The transgene encoded R55VH regions had overall levels, and frequencies of mutation clustered to mutational hot spots (Rogozin and Kolchanov, 1992; Shapiro et al., 1999) comparable to those in endogenously encoded canonical VH regions obtained from the same mice. All of the V␬ genes coexpressed with the transgene-encoded VH region were canonical and displayed levels of somatic mutation similar to the canonical V␬ genes coexpressed with VH regions encoded by endogenous IgH loci. Nearly half of the transgene-encoded VH regions had the Thr58→Ile mutation, previously shown to increase the Ars affinity of canonical V regions (Casson and Manser, 1995; Sharon et al., 1989) (data not shown). Affinity maturation was confirmed by fluorescence quenching analysis of BVER mAbs containing the trans-

genic R55VH region using Ars-N-acetyl-L-tyrosine (Table 2). The average KA of these mAbs was 1.6 ⫻ 107 M⫺1, substantially higher than the KA of unmutated canonical mAbs such as 36-65, as well as R55/223 (Table 2). In total, all of the transgene-encoded mAbs had increased affinity for Ars, while 8 out of 13 had at least 10-fold increased affinity. This increase in KA is characteristic of affinity-matured, hypermutated canonical antibodies expressed during an Ars immune response in nontransgenic A strain mice (Manser, 1991). Canonical mAbs from BVER55 Mice Display Reduced Binding to Autoantigens To investigate whether the autoantigen binding capabilities conferred by the R55 mutation were attenuated in the canonical BCRs expressed by B cells that had undergone Ars-driven somatic mutation and selection in BVER55 mice, R55VH transgene-encoded mAbs obtained from this line were tested for ssDNA, dsDNA, and


Table 2. Ars Affinities of Purified mAbs Containing the Transgenic VH Region mAb

Affinity (KA ⫽ M⫺1)a

Unmutated Canonical Control 36-65 R55 Mutated Canonical Control R55/223 mAbs from BVER55 Secondary Fusions 11-C4 11-C7 11-A4 22-5E12-B7 22-1E12-F6 22-4B10-C8 22-3B9-E6 23-1G5-F9 24-4A11-D6 24-3C12-D3 24-1H9-D6 24-1E7-F1 24-1F9-F3

4.7 ⫻ 105 4.4 ⫻ 105 1.6 ⫻ 107 6.1 ⫻ 105, 4.7 1.1 ⫻ 107, 3.0 6.0 ⫻ 106 1.5 ⫻ 106, 9.0 ⱖ1.3 ⫻ 108 ⱖ1.3 ⫻ 108 8.7 ⫻ 105, 7.8 6.0 ⫻ 106, 7.0 8.7 ⫻ 105, 6.1 2.4 ⫻ 106, 3.3 1.3 ⫻ 108 8.7 ⫻ 105, 1.6

⫻ 105 ⫻ 107 ⫻ 105

⫻ ⫻ ⫻ ⫻

105 106 105 106

⫻ 106

a

Multiple KA values were each obtained from an independent experiment.

chromatin binding (Figure 5). Most of the transgeneencoded mAbs retained their ability to bind ssDNA and dsDNA. However, all displayed reduced binding to these autoantigens as compared to the R55/233 mAb (Figure 5). Moreover, all of the mAbs exhibited very weak or undetectable binding to chromatin (Figure 5). ANA reactivities of the mAbs containing the transgenic R55VH were compared to PL9-6 and 36-65, which are ANA positive and negative under the conditions used, respectively (data not shown). Only 1 out of the 13 mAbs gave a detectable signal in these assays. However, this

mAb gave rise to mainly perinuclear and weak nuclear staining, which excluded some nuclear structures, and this staining was less intense than that conferred by the R55 mutation alone, even at high concentrations of antibody.

Discussion Several studies have suggested that self-antigen-regulated B cells may be capable of participating in foreign antigen-driven immune responses (Cooke et al., 1994; Noorchashm et al., 1999; Sater et al., 1998; Seo et al., 2002), raising the possibility that autoreactive memory B cells could develop during such responses. Our data show that although canonical R55VH transgene-expressing B cells colonize follicles and can participate in the foreign antigen-induced GC response, they appear incapable of entering the memory compartment. Thus, the data suggest that negative selection by self-antigen is dominant over positive selection for increased affinity for foreign antigen during the BCR mutation-selection process. Furthermore, enforced expression of Bcl-2 perturbed the negative selection of B cell clones expressing the transgenic R55VH region such that their BCRs were readily detected in the Ars-induced memory compartment, but these BCRs had altered autoreactivities. All of the transgene-encoded canonical mAbs isolated from BVER55 mice bound less well to nucleic acid autoantigens than the unmutated canonical R55 V region. Thus, the ability of Bcl-2 to modify the negative selection of B cells with certain autoreactivities and not others suggests a hierarchy of peripheral negative selection by

Figure 5. Relative Binding of BVER55 Secondary mAbs to Chromatin, ssDNA, and dsDNA Purified transgene-encoded mAbs derived from secondary anti-Ars responses of four BVER55 mice were evaluated for relative binding to these ligands by ELISA. These binding activities were compared to those of PL9-6 (filled triangles, dashed line), R55/223 (filled diamonds, solid line), and 36-65/223 (filled squares, dashed line).

Immunity 324

autoantigen(s) during the mutation-selection pathway leading to memory B cell development. In accordance with previous studies (Goodnow et al., 1988; Mandik-Nayak et al., 1997), our finding in HKIR mice that surface IgM and IgD levels on canonical R55VH preimmune B cells are reduced indicates that such cells are subjected to self-antigen-mediated regulation, i.e., they are not ignorant of self-antigens. In agreement with this idea, Ars-KLH immunization induced canonical R55VH-expressing B cells to enter GCs, but none of the AFCs observed during this response were E4⫹. While further studies will be required to completely elucidate the phenotypic status and functional potential of preimmune canonical R55VH B cells, these observations support the conclusion that self-antigen-regulated B cells can enter the memory B cell specificity maturation pathway when stimulated by foreign antigen. A previous study of B cells in a conventional Ig transgenic line of mice expressing an unmutated, canonical VH gene and a heavily mutated, canonical light chain gene revealed downregulation of sIgM but elevated levels of sIgD and that these cells were incapable of responding to Ars immunization in vivo or BCR engagement in vitro (Benschop et al., 2001). Like canonical R55 V regions, the particular VH:VL combination expressed in these mice binds Ars with low affinity, but it differs in binding DNA poorly. The reasons for the distinct behaviors of B cells expressing these two forms of canonical V regions will require further analysis. However, it is likely that differences in the specificities and levels of expression of their BCRs are major contributing factors (Heltemes and Manser, 2002). One explanation for the dominance of negative selection by autoantigen over positive selection by foreign antigen during specificity maturation may be provided by the order of these events. Positive selection of B cells expressing high-affinity BCRs is argued to be mediated via the interaction of the BCR with immune complexes (IC) deposited on follicular dendritic cells (FDCs), while negative selection is suggested to occur through interaction with CD4⫹ T cells (MacLennan, 1994). Given these ideas, B cells given a survival signal following interaction with IC on FDCs may present the antigen acquired from these IC to CD4⫹ T cells (MacLennan, 1994). Self-reactive B cells that attempt to present autoantigen or are precluded from presenting foreign antigen to tolerant CD4⫹ T cells are negatively selected via altered signaling in the absence of costimulation (Goodnow et al., 1995) or by Fas-mediated apoptosis (Rathmell et al., 1995). Thus, positive selection via IC on FDCs may occur prior to negative selection via T cell interaction, and accordingly, the dominance of negative selection over positive selection may naturally follow. The reduced but not ablated autoreactivities of canonical R55VH-encoded mAbs isolated from BVER mice may be attributed to the difference in affinities of the transgenic R55VH region for different forms of nucleic acid autoantigens. If developing memory B cells expressing the transgenic R55VH region have a higher affinity for chromatin than free DNA, binding to chromatin may result both in more extensive crosslinking of the BCR than when DNA is bound and in the transmission of a death signal to the cell. In addition, free DNA may be scarce as compared to chromatin in vivo. The combination of

these two factors could result in chromatin being a stronger tolerogen. In general, ubiquitous intracellular autoantigens such as chromatin may be readily available in the GC, perhaps on the surface of apoptotic cells (Rosen et al., 1995), but other self-antigens may be less accessible to hypermutating B cells. Studies by Reed et al. (2000) on transgenic mice expressing influenza hemagglutinin (HA) as a neo-self-antigen suggested that anti-HA B cells were not subjected to a peripheral tolerance checkpoint during memory B cell genesis. The above arguments would predict that in these mice HA is not expressed at significant levels in GCs. Since canonical R55VH transgene-expressing-B cells were detected in GCs but appeared to be grossly underrepresented in the memory compartment in the absence of enforced Bcl-2 expression, it is possible that the selfreactivity conferred by R55 could not be completely revised via V gene hypermutation. Indeed, all of the R55VH genes expressed by the BVER55 hybridomas had an intact R55 codon, demonstrating that the alterations in the autospecificities of the encoded mAbs resulted from second site mutations. Therefore, the R55 codon may be a hypermutation cold spot (Radmacher et al., 1998). Consequently, the inability of second site mutations to ablate the autoreactivity conferred by the R55 mutation may have resulted in the deletion of nearly all transgene-expressing B cells within the GC in mice not expressing the Bcl-2 transgene. This may account for our observation that GCs containing canonical R55VHexpressing B cells were rare in Ars-KLH-immunized VR55 and VER55 mice. Such GCs could be easily observed in immunized HKIR chimeric mice, but this probably resulted from the elevation of the frequency of canonical R55VH-expressing precursors. Accordingly, the one canonical R55VH transgene-encoded mAb identified in a total of 163 canonical hybridomas isolated from VR55 and VER55 mice lacked DNA reactivity and was a somatically mutated IgG. Collectively, these data support the idea that memory B cell precursors are susceptible to a stringent peripheral tolerance mechanism(s) operative in the GC (Han et al., 1995; Linton et al., 1991; Pulendran et al., 1995; Shokat and Goodnow, 1995). Since autoantibodies with high affinity for native nuclear antigens such as chromatin are characteristic of many systemic autoimmune diseases, the need for negative regulation of such specificities seems obvious. In contrast, whether B cells with reactivity for naked DNA pose a pathologic threat has been less clear. Nonetheless, although DNA reactive B cells have been demonstrated to be common residents of the periphery of normal individuals (Limpanasithikul et al., 1995; Rousseau et al., 1989) and to arise as a consequence of somatic hypermutation (Casson and Manser, 1995; Diamond et al., 1992), most data are consistent with those presented here in supporting the idea that such cells do not enter the memory compartment under normal conditions (Borretzen et al., 1994; Hande et al., 1998; Naparstek et al., 1986). The physiological significance of the regulation of B cell DNA reactivity has been suggested by recent studies of DNase I-deficient mice. These mice develop high titers of pathogenic autoantibodies, apparently due to altered clearance of extracellular DNA (Napirei et al., 2000; Walport, 2000). B cell tolerance begins in the bone marrow, where


immature B cells with BCRs capable of binding selfantigen are eliminated by clonal deletion (Chen et al., 1995; Nemazee and Burki, 1989) or undergo receptor revision (Gay et al., 1993; Tiegs et al., 1993). Subsequently, autoreactive B cells that enter the periphery are either clonally ignorant (Goodnow, 1992; Nossal, 1993), rendered anergic (Erikson et al., 1991; Goodnow et al., 1988), or deleted by self-antigen (Monroe, 1998; Rathmell et al., 1995). If autoreactive memory B cells do develop, they may be subjected to tolerance mechanisms that preclude their differentiation to AFC (Hande et al., 1998; Walker and Weigle, 1985). The data presented here demonstrate that B cells that are strongly reactive with intracellular autoantigens and are recruited to participate in a TD immune response are subjected to peripheral tolerance mechanisms during the mutationselection memory B cell pathway. Enforced expression of Bcl-2 alters this process and permits B cell clones with certain autoreactivities to enter the memory compartment. Collectively, these data support the existence of hierarchical peripheral tolerance mechanisms operative during multiple stages of both primary and secondary B cell development. Experimental Procedures Transgenic, Knockin, and Chimeric Mice The generation of the pVHXbaRI/R55 and pVHXbaI3⬘Enh/R55 vectors for the construction of the VR55 and VER55 transgenic mice, respectively, was similar to that described (Giusti et al., 1992). Both contain a H chain VHDJH exon from the 36-65 hybridoma (Marshak-Rothstein et al., 1980) with an AAT (Asn) → CGA (Arg) mutation at codon 55, the rare V-D and D-J junctions of 36-65, 5⬘ flanking VH leader and promoter elements, and plasmid flanking DNA. The rare junctions confer reactivity with the anti-idiotypic antibody 107 (Giusti et al., 1992; Weissman et al., 1985) allowing for distinction between endogenous canonical VH regions and transgene-encoded VH regions. Both constructs lack any switch or constant region DNA. pVHXbaI3⬘Enh/ R55 also contains the 3⬘ H chain enhancer (hs 1,2) and ⵑ500 bp of SV40 intronic sequence. The resulting lines of mice were backcrossed to A strain mice for at least five generations. To obtain the BVER55 mouse line, the VER5589 line was crossed to A.Bcl-2 transgenic mice (Hande et al., 1998; McDonnell et al., 1989). The pKI-36-65Arg vector used to create the HKIR line of VH knockin mice was generated using the pKO Scrambler series from Stratagene (La Jolla, CA). Restriction fragments containing the Diptheria toxin gene (pKO SelectDT) and the neomycin gene with flanking loxP sites (Osdupl; provided by Dr. Oliver Smithies) were cloned into the pKO Scrambler V908 vector. The Balb/c DQ52 gene and flanking sequence was excised as a 2.3 kb fragment from a plasmid vector (Dirkes et al., 1994), and a 1 kb region containing the 129/ SvJ IgH intronic enhancer and 5⬘ flanking sequence (3⬘ recombination site) was PCR amplified, and both were cloned into the vector. Finally, the 36-65 VDJ fragment was PCR amplified using primers that introduced the position 55 AAT → CGA (R) change as was previously described (Casson and Manser, 1995) and was cloned into the vector. NotI linearized vector was transfected into a 129/ SvJ ES cell line obtained from Incyte Genomics (St. Louis, MO). Subclones containing appropriately targeted alleles were identified by Southern blot assaying for the presence of a 2.1 kb StuI fragment from the targeted allele as compared to a 4.7 kb fragment derived from the wild-type allele using the J14C-S probe (see Figure 2). Genomic DNA was prepared from ES cells as described by Stratagene (La Jolla, CA). ES cell subclones were microinjected into C57BL/6 blastocysts, and the resulting chimeras were crossed to C57BL/6 mice. Agouti offspring were screened by Southern blot hybridization of StuI digested tail DNA with the J14 C-S probe. For some experiments, RBC-depleted HKIR splenocytes were transferred to unirradiated, syngeneic littermates (2 ⫻ 106 cells per

recipient) via tail vein injection. Twelve hours later the chimeric mice were immunized with 100 ␮g of Ars-KLH in alum IP. At various times thereafter, the mice were killed, and spleens were taken and processed for histology. Flow Cytometry Single-cell suspensions were prepared from lymphoid organs of 8- to 10-week old, naive HKIR mice and age-matched transgenenegative littermates. Cells were stained with different combinations of the following antibodies: ␣-IgM-FITC (Jackson ImmunoResearch; West Grove, PA), ␣-IgD-PE (clone 11-26; Southern Biotechnology Associates), ␣-IgMa-FITC (clone DS-1), ␣-IgMb-PE (clone AF6-78), ␣-CD21/35-FITC (clone 7G6), ␣-CD23-PE (clone B3B4), ␣-CD24-PE (HSA, clone M1/69), ␣-CD45R-biotin, -FITC, and -PE (B220, clone RA3-6B2) (biotin; eBioscience, San Diego, CA), or ␣-idiotypic biotinylated mAb E4. All antibodies were obtained from BD Biosciences, Pharmingen (San Diego, CA) unless otherwise indicated. Anti-idiotypic antibodies were purified and biotinylated using standard methods. CyChrome (BD Biosciences, Pharmingen, San Diego, CA) was used as a second step reagent. Cells were fixed in 1% paraformaldehyde, assayed on a Coulter Epics Elite, and data analyzed using FLOWJO software (Treestar; San Carlos, CA). Hybridomas VR55, VER55, and BVER55 mice at least 8 weeks of age were primed IP with 100 ␮g of Ars-KLH emulsified in complete Freund’s adjuvant (CFA). Some mice were then boosted IP with 50 ␮g Ars-KLH in PBS at least 1 month after primary immunization for secondary responses. For tertiary responses, mice were boosted IP with 50 ␮g Ars-KLH in PBS at least 1 month after primary immunization and once again at least 1 month later. Mice were sacrificed at various time points during primary responses (days 4, 7, 10, 12, and 15) and 3 days after the final boost for secondary and tertiary responses. For hyperimmunizations, mice were primed with 100 ␮g Ars-KLH in CFA, boosted a week later with 50 ␮g Ars-KLH in PBS, and then boosted three more times with this amount of antigen, once every other day. Spleen cells were used to generate hybridomas as described (Manser, 1989) using the Sp2/0 fusion partner. Hybridoma supernatants were screened for the presence of canonical antibodies via an anti-idiotypic (E4) ELISA (Casson and Manser, 1995; Hande et al., 1998). E4⫹ hybridomas were subcloned by limiting dilution. Purification of mAbs, mAb Binding Assays, and Determination of mAb Affinity mAbs were purified from hybridoma supernatants using protein G chromatography (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s suggestions. E4 and DNA reactivities were measured by solid-phase ELISAs as described before (Casson and Manser, 1995; Hande et al., 1998). Chromatin reactivity was measured by ELISA as described elsewhere (Fowler and Cheng, 1983). Bound antibodies were elaborated with a biotinylated rat anti-mouse ␬ mAb followed by streptavidin alkaline phosphatase (AP) (Southern Biotechnology Associates, Inc., Birmingham, AL) and PNPP (Sigma Chemical Diagnostics, St. Louis, MO). The affinities of purified antibodies were determined by fluorescence quenching using ArsN-acetyl-L-tyrosine as previously described (Hande et al., 1998; Rothstein and Gefter, 1983) and a Perkin-Elmer (model LS-50) spectrophotofluorimeter. Immunohistochemistry Spleens were removed, flash frozen, sectioned, and fixed as previously described (Jacob et al., 1991). Five to six micrometer frozen sections were thawed, rehydrated, and stained as before (Hande et al., 1998; Jacob et al., 1991). To identify canonical antibodyexpressing cells, sections were incubated with either biotinylated E4 or 107, washed in TBS/BSA, and then incubated with Streptavidin-AP (Southern Biotechnology Associates, Inc. Birmingham, AL). GCs and CD4⫹ T cells were identified with PNA (E-Y Laboratories, San Mateo, CA) and the mAb GK1.5 coupled to horseradish peroxidase (HRP), respectively. Bound AP and HRP activities were visualized using Napthol AS-MX/Fast Blue BB Base and 3-aminoethylcarbazole, respectively.

Immunity 326

Immune Complex Deposition SCID mice injected with 3 ⫻ 106 transfectoma cells were sacrificed approximately 2 weeks later. Kidneys were flash frozen, and 6 ␮M sections were prepared as above. Immune complex deposition was revealed with a FITC-labeled goat anti-mouse ␬ mAb (Southern Biotechnology Associates Inc., Birmingham, AL). ANA Staining ANA reactivity of purified mAbs was determined using human epithelioid Hep-2 cells on prepared slides (Antibodies Incorporated, Davis, CA) according to the manufacturer’s instructions. Slides were stained with 20 ␮l of 10 or 25 ␮g/ml purified mAb. Bound antibodies were detected with a FITC-labeled goat anti-mouse ␬ antibody (Southern Biotechnology Associates, Birmingham, AL). Construction of the VHR55 Expression Vector and Electroporation of 36-65L Cells A restriction fragment containing the R55VH region was isolated from an intermediate vector and ligated into the #223␥2b expression vector (Gillies et al., 1983). A control #223␥2b expression vector with the 36-65 VH (36-65/223) was also constructed using the same approach. Plasmids were then electroporated into the 36-65L cell line (Sharon et al., 1984) as described (Sharon et al., 1989) with minor modifications. Following 24–48 hr, transfectants were selected using mycophenolic acid for 7–10 days, and expression of secreted antibody was determined by an Ars ELISA (Casson and Manser, 1995; Hande et al., 1998). Positive clones were expanded, and their mAbs were purified as described above. Acknowledgments We thank Dr. Behnaz Parhami-Seren for her assistance in constructing the 36-65/223 and R55/223 mAbs, Dr. Marc Monestier for providing the PL9-6 mAb and hybridoma, Dr. Shailaja Hande for making the transgene constructs used to generate the VR55 and VER55 mice, and Dr. Lesley Lock and the other members of the KCC transgenic/knockout facility. We also thank William Monsell and Kate Dugan for their technical assistance and all other members of the Manser laboratory for their indirect contributions to this work. This study was supported by an NIH grant to T.M. (AI38965). E.N. was supported by NIH training grant AI-07492, and L.H. by NIH training grant CA-72318. Received: June 26, 2001 Revised: August 8, 2002 References

Scharff, M.D. (1992). The role of somatic mutation in the pathogenic anti-DNA response. Annu. Rev. Immunol. 10, 731–757. Dirkes, G., Kohler, G., and Kottmann, A.H. (1994). Sequence and structure of the mouse IgH DQ52 5⬘ region. Immunogenetics 40, 379–390. Erikson, J., Radic, M.Z., Camper, S.A., Hardy, R.R., Carmack, C., and Weigert, M. (1991). Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature 349, 331–334. Fowler, E., and Cheng, N. (1983). Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components. J. Immunol. Methods 62, 297–303. Gay, D., Saunders, T., Camper, S., and Weigert, M. (1993). Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177, 999–1008. Gillies, S.D., Morrison, S.L., Oi, V.T., and Tonegawa, S. (1983). A tissue specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell 33, 717–730. Giusti, A.M., Coffee, R., and Manser, T. (1992). Somatic recombination of heavy chain variable region transgenes with the endogenous immunoglobulin heavy chain locus in mice. Proc. Natl. Acad. Sci. USA 89, 10321–10325. Goodnow, C.C. (1992). Transgenic mice and analysis of B-cell tolerance. Annu. Rev. Immunol. 10, 489–514. Goodnow, C.C., Crosbie, J., Adelstein, S., Lavoie, T.B., Smith-Gill, S.J., Brink, R.A., Pritchard-Briscoe, H., Wotherspoon, J.S., Loblay, R.H., Raphael, K., et al. (1988). Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334, 676–682. Goodnow, C.C., Cyster, J.G., Hartley, S.B., Bell, S.E., Cooke, M.P., Healy, J.I., Akkaraju, S., Rathmell, J.C., Pogue, S.L., and Shokat, K.P. (1995). Self-tolerance checkpoints in B lymphocyte development. Adv. Immunol. 59, 279–368. Han, S., Zheng, B., Porto, J.D., and Kelsoe, G. (1995). In situ studies of the primary response to (4-hydroxy-3-nitrophenyl)acetyl. IV. Affinity-dependent, antigen-driven B cell apoptosis in germinal centers as a mechanism for maintaining self-tolerance. J. Exp. Med. 182, 1635–1644. Hande, S., and Manser, T. (1998). Single amino acid substitutions in Vh CDR2 are sufficient to generate or enhance the specificity of two forms of an anti-arsonate antibody variable region for DNA. Mol. Immunol. 34, 1281–1290. Hande, S., Notidis, E., and Manser, T. (1998). Bcl-2 obstructs negative selection of autoreactive, hypermutated antibody V regions during memory B cell development. Immunity 8, 189–198.

Benschop, R.J., Aviszus, K., Zhang, X., Manser, T., Cambier, J.C., and Wysocki, L.J. (2001). Activation and anergy in bone marrow B cells of a novel immunoglobulin transgenic mouse that is both hapten specific and autoreactive. Immunity 14, 33–43.

Heltemes, L.M., and Manser, T. (2002). Level of B cell antigen receptor surface expression influences both positive and negative selection of B cells during primary development. J. Immunol. 169, 1283– 1292.

Borretzen, M., Randen, I., Zdarsky, E., Forre, O., Natvig, J.B., and Thompson, K. (1994). Control of autoantibody affinity by selection against amino acid replacements in the complementarity-determining regions. Proc. Natl. Acad. Sci. USA 91, 12917–12921.

Hiernaux, J., Slaoui, M., Leo, O., Moser, M., Franssen, J.D., and Urbain, J. (1983). Studies of the arsonate system using monoclonal antibodies. Ann. NY Acad. Sci. 418, 9–15.

Casson, L.P., and Manser, T. (1995). Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies for both cognate antigen and autoantigen. J. Exp. Med. 182, 743–750. Chen, C., Nagy, Z., Radic, M.Z., Hardy, R.R., Huszar, D., Camper, S.A., and Weigert, M. (1995). The site and stage of anti-DNA B-cell deletion. Nature 373, 252–255. Cooke, M.P., Heath, A.W., Shokat, K.M., Zeng, Y., Finkelman, F.D., Linsley, P.S., Howard, M., and Goodnow, C.C. (1994). Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells. J. Exp. Med. 179, 425–438.

Jacob, J., Kassir, R., and Kelsoe, G. (1991). In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173, 1165–1175. Karlsen, A.E., and Dyrberg, T. (1998). Molecular mimicry between non-self, modified self and self in autoimmunity. Semin. Immunol. 10, 25–34. Krishnan, M.R., and Marion, T.N. (1993). Structural similarity of antibody variable regions from immune and autoimmune anti-DNA antibodies. J. Immunol. 150, 4948–4957. Limpanasithikul, W., Ray, S., and Diamond, B. (1995). Cross-reactive antibodies have both protective and pathogenic potential. J. Immunol. 155, 967–973.

Cyster, J.G., Hartley, S.B., and Goodnow, C.C. (1994). Competition for follicular niches excludes self-reactive cells from the recirculating B-cell repertoire. Nature 371, 389–395.

Linton, P.-J., Rudie, A., and Klinman, N.R. (1991). Tolerance susceptibility of newly generating memory B cells. J. Immunol. 146, 4099– 4104.

Diamond, B., Katz, J.B., Paul, E., Aranow, C., Lustgarten, D., and

Litzenburger, T., Fassler, R., Bauer, J., Lassmann, H., Linington,


C., Wekerle, H., and Iglesias, A. (1998). B lymphocytes producing demyelinating autoantibodies: development and function in genetargeted transgenic mice. J. Exp. Med. 188, 169–180. Losman, M.J., Fasy, T.M., Novick, K.E., and Monestier, M. (1993). Relationships among antinuclear antibodies from autoimmune MRL mice reacting with histone H2A-H2B dimers and DNA. Int. Immunol. 5, 513–523. MacLennan, I.C.M. (1994). Germinal centers. Annu. Rev. Immunol. 12, 117–139. Mandik-Nayak, L., Noorchashm, H., Eaton, A., and Erikson, J. (1997). Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle. J. Exp. Med. 186, 1257–1267. Manser, T. (1989). Evolution of antibody structure during the immune response. J. Exp. Med. 170, 1211–1230. Manser, T. (1991). Regulation, timing and mechanism of antibody V-gene somatic hypermutation: lessons from the arsonate system. In Somatic Hypermutation in V-Regions, E.J. Steele, ed. (Boca Raton, FL: CRC Press), pp. 42–54. Manser, T., and Gefter, M.L. (1986). The molecular evolution of the immune response: idiotype specific suppression indicates that B cells express germline encoded V genes prior to antigenic stimulation. Eur. J. Immunol. 16, 1439–1447. Manser, T., Wysocki, L.J., Margolies, M.N., and Gefter, M.L. (1987). Evolution of antibody variable region structure during the immune response. Immunol. Rev. 96, 141–162. Marshak-Rothstein, A., Siekevitz, M., Margolies, M.N., MudgettHunter, M., and Gefter, M.L. (1980). Hybridoma proteins expressing the predominant idiotype of the anti-azophenylarsonate response of A/J mice. Proc. Natl. Acad. Sci. USA 77, 1120–1124.

Oldstone, M.B. (1998). Molecular mimicry and immune-mediated diseases. FASEB J. 12, 1255–1265. Parker, D.C., and Eynon, E.E. (1991). Antigen presentation in acquired immunological tolerance. FASEB J. 5, 2777–2784. Pulendran, B., Kannourakis, G., Nouri, S., Smith, K.G.C., and Nossal, G.J.V. (1995). Soluble antigen can cause enhanced apoptosis of germinal-centre B cells. Nature 375, 331–334. Radmacher, M.D., Kelsoe, G., and Kepler, T.B. (1998). Predicted and inferred waiting times for key mutations in the germinal center reaaction: evidence for stochasticity in selection. Immunol. Cell Biol. 76, 373–381. Rathmell, J.C., Cooke, M.P., Ho, W.Y., Grein, J., Townsend, S.E., Davis, M.M., and Goodnow, C.C. (1995). CD95 (FAS)-dependent elimination of self-reactive B cells upon interaction with CD4⫹ T cells. Nature 376, 181–184. Reed, A.J., Riley, M.P., and Caton, A.J. (2000). Virus-induced maturation and activation of autoreactive memory B cells. J. Exp. Med. 192, 1763–1774. Rogozin, I.B., and Kolchanov, N.A. (1992). Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequences on mutagenesis. Biochim. Biophys. Acta 1171, 11–18. Rosen, A., Casciola-Rosen, L., and Ahearn, J. (1995). Novel packages of viral and self-antigens are generated during apoptosis. J. Exp. Med. 181, 1557–1561. Rothstein, T.L., and Gefter, M.L. (1983). Affinity analysis of idiotypepositive and idiotype negative Ars-binding hybridoma proteins and Ars-immune sera. Mol. Immunol. 20, 161–168. Rousseau, P.G., Mallett, C.P., and Smith-Gill, S.J. (1989). A substantial proportion of the adult BALB/c available B cell repertoire consists of multireactive B cells. Mol. Immunol. 26, 993–1006.

McDonnell, T.J., Deane, N., Platt, F.M., Nunez, G., Jaeger, U., McKearn, J.P., and Korsmeyer, S. (1989). Bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell 57, 79–88.

Sater, R.A., Sandel, P.C., and Monroe, J.G. (1998). B cell receptorinduced apoptosis in primary transitional murine B cells: signaling requirements and modulation by T cell help. Int. Immunol. 10, 1673– 1682.

Moens, U., Seternes, O.-M., Hey, A.W., Silsand, Y., Traavik, T., Johansen, B., and Rekvig, O.P. (1995). In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosusrelated autoimmunity to doubled-stranded DNA and histones. Proc. Natl. Acad. Sci. USA 92, 12393–12397.

Seo, S., Fields, M.L., Buckler, J.L., Reed, A.J., Mandik-Nayak, L., Nish, S.A., Noelle, R.J., Turka, L.A., Finkelman, F.D., Caton, A.J., and Erikson, J. (2002). The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity 16, 535–546.

Monroe, J.G. (1998). Antigen receptor-initiated signals for B cell development and selection. Immunol. Res. 17, 155–162.

Shapiro, G.S., Aviszus, K., Ikle, D., and Wysocki, L.J. (1999). Predicting regional mutability in antibody V genes based solely on diand trinucleotide sequence composition. J. Immunol. 163, 259–268.

Murakami, M., Tsubata, T., Okamoto, M., Shimizu, A., Kumagai, S., Imura, H., and Honjo, T. (1992). Antigen-induced apoptotic death of Ly-1 B cells responsible for autoimmune disease in transgenic mice. Nature 357, 77–80. Naparstek, Y., Andre-Schwartz, J., Manser, T., Wysocki, L.J., Breitman, L., Stollar, B.D., Gefter, M.L., and Schwartz, R.S. (1986). A single germline VH gene segment of normal A/J mice encodes autoantibodies characteristic of systemic lupus erythematosus. J. Exp. Med. 164, 614–626. Napirei, M., Karsunky, H., Zevnik, B., Stephan, H., Mannherz, H.G., and Moroy, T. (2000). Features of systemic lupus erythematosus in DNase 1-deficient mice. Nat. Genet. 25, 177–181.

Sharon, J., Gefter, M.L., Manser, T., Morrison, S.L., Oi, V.T., and Ptashne, M. (1984). Expression of a VHC␬ chimeric protein in mouse myeloma cells. Nature 309, 364–367. Sharon, J., Gefter, M.L., Wysocki, L.J., and Margolies, M.N. (1989). Recurrent somatic mutations in mouse antibodies to p-azophenylarsonate increase affinity for hapten. J. Immunol. 142, 596–601. Shokat, K.M., and Goodnow, C.C. (1995). Antigen-induced B-cell death and elimination during germinal-centre immune apoptosis. Nature 375, 334–338. Tiegs, S.L., Russell, D.M., and Nemazee, D. (1993). Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 177, 1009–1020.

Nemazee, D.A., and Burki, K. (1989). Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature 337, 562–566.

Vora, K.A., Tumas-Brundage, K., and Manser, T. (1999). Contrasting the in situ behavior of a memory B cell clone during primary and seconday immune responses. J. Immunol. 163, 4315–4327.

Nguyen, K.-A.T., Mandik, L., Bui, A., Kavaler, J., Norvell, A., Monroe, J.G., Roark, J.H., and Erikson, J. (1997). Characterization of antisingle-stranded DNA B cells in a non-autoimmune background. J. Immunol. 159, 2633–2644.

Walker, S.M., and Weigle, W.O. (1985). Primed lymphoid cell tolerance. II. In vivo tolerization of highly tolerogen-sensitive haptenprimed, potentially IgG-producing B cells. Cell. Immunol. 90, 331–338.

Noorchashm, H., Bui, A., Li, H.-L., Eaton, A., Mandik-Nayak, L., Sokol, C., Potts, K.M., Pure, E., and Erikson, J. (1999). Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process. Int. Immunol. 11, 765–776.

Walport, M.J. (2000). Lupus, DNase and defective disposal of cellular debris. Nat. Genet. 25, 135–136.

Nossal, G.J.V. (1993). Tolerance and ways to break it. Ann. NY Acad. Sci. 690, 34–41. Notidis, E., Hande, S., and Manser, T. (2001). Enforced expression of Bcl-2 selectively perturbs negative selection of dual reactive antibodies. Dev. Immunol. 8, 223–234.

Weissman, D., Rothstein, T.L., and Marshak-Rothstein, A. (1985). A rapid method for comparing monoclonal antibodies by limited proteolysis and electrophoresis. Hybridoma 4, 329–337.