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The Journal of Immunology, 2004, 173: 5485-5494.
Copyright © 2004 by The American Association of Immunologists

Specificity-Based Negative Selection of Autoreactive B Cells during Memory Formation1

Heath M. Guay, Laura Panarey, Amy J. Reed and Andrew J. Caton2

The Wistar Institute, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Autoreactive B cells are not completely purged from the primary B cell repertoire, and whether they can be prevented from maturation into memory B cells has been uncertain. We show here that a population of B cells that dominates primary immune responses of BALB/c mice to influenza virus A/PR/8/34 hemagglutinin (HA) are negatively selected in transgenic mice expressing PR8 HA as an abundant membrane-bound Ag (HACII mice). However, a separate population of B cells that contains precursors of memory B cells is activated by PR8 virus immunization and is subsequently negatively selected during the formation of the memory response. Negative selection of PR8 HA-specific B cells altered the specificity of the memory B cell response to a mutant virus containing a single amino acid substitution in a B cell epitope. Strikingly, this skewed reactivity resulted from an increase in the formation of memory B cells directed to non-self-epitopes on the mutant virus, which increased 8-fold in HACII mice relative to nontransgenic mice and precisely compensated for the absence of autoreactive PR8 HA-specific memory B cells. Negative selection of PR8 HA-specific B cells was a dominant process, since B cells from HACII mice could induce negative selection of PR8 HA-specific B cells from BALB/c mice. Lastly, HA-specific memory responses were unaffected by self-tolerance in another lineage of HA-transgenic mice (HA104 mice), indicating that the amount and/or cell type in which self-Ags are expressed can determine their ability to prevent autoreactive memory B cell formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
B cell repertoire formation occurs in distinct phases and in discrete anatomical sites. The primary B cell repertoire is formed mainly in the bone marrow (BM)3 through the rearrangement of Ig variable region genes, leading to the development of B cells expressing functional Ig molecules with sufficient diversity to recognize an unknown universe of pathogens (1). The memory B cell repertoire, in contrast, is formed principally in germinal centers that develop in the spleen and lymph node in response to signals from inciting agents, including CD4+ T cells (2, 3, 4). Within germinal centers, a subset of the Ag-specific B cells activated from the primary repertoire undergo somatic hypermutation and a complex selection process termed affinity maturation that is characterized by the consistent expansion of rare variants possessing higher affinity and enhanced specificity for the immunizing agent (2, 3, 4). Somatically mutated autoantibodies are also characteristic of some autoimmune diseases (5, 6, 7). Yet whether and why these cells evade mechanisms that might actively eliminate autoreactive B cells from the memory repertoire is not understood.

Studies of Ab responses to variants of self-Ags have shown that the specificity of the memory B cell repertoire can be skewed away from reactivity with self-Ags (8, 9, 10). These studies indicated that B cell tolerance induction occurs and can shape the specificity of memory responses, although whether this reflected processes occurring during primary or memory formation (or both) was not known. Mice expressing defined Ig specificities either as transgenes or following targeted integration into Ig loci have been used to show that B cells expressing autoreactive specificities can be negatively selected from the primary repertoire, either through deletion (11, 12) or receptor editing (13, 14). In other cases, B cells expressing autoreactive specificities were found to persist in the preimmune repertoire despite their specificity for a self-Ag, and may be actively regulated by anergy induction (15, 16) or follicular exclusion (17, 18). However, there is also evidence that these autoreactive B cells can be recruited into Ag-driven immune responses if provided with CD4+ T cell help (19, 20, 21). Indirect evidence that autoreactive B cells may be subjected to tolerance induction during memory formation was obtained by showing that large doses of soluble Ag can induce apoptosis of germinal center B cells (22, 23). Also, introduction of a Bcl-2 transgene allowed B cells with dual specificity for a foreign and self-Ag to be recovered from germinal center B cells (24, 25), providing further evidence that apoptosis may play a role in preventing autoreactive memory B cell formation. Nevertheless, direct evidence that autoreactive B cells can be subjected to negative selection during memory formation based on their specificity for a self-Ag has been sparse.

We have been examining the extent and specificity with which autoreactive B cells are negatively selected from primary and memory B cell repertoires using the influenza virus A/PR/8/34 hemagglutinin (PR8 HA) as a model self-Ag. We previously examined tolerance induction in HA104 mice, in which PR8 HA expression is driven by the SV40 early region promoter/enhancer, and showed that a population of B cells that dominates primary responses to PR8 HA in BALB/c mice is subjected to negative selection in HA104 mice (26). However, another population, which participates in both primary and memory B cell responses of BALB/c mice, evaded negative selection from the primary B cell repertoire of HA104 mice (26), and memory B cell responses to the PR8 HA were unaffected by tolerance induction (27). In this report, we have analyzed transgenic mice that express PR8 HA as an abundant membrane-bound Ag driven by a MHC class II promoter (HACII mice) (28). In this case too, a population of autoreactive PR8 HA-specific B cells can be activated by primary virus immunization, but unlike HA104 mice, these autoreactive PR8 HA-specific B cells are subjected to negative selection during memory formation in HACII mice. These studies provide direct evidence that specificity for self-Ag can prevent autoreactive B cells that evade negative selection from the primary repertoire from maturing into memory B cells. The findings further show that the ability of self-Ags to impose this negative selection can be affected by their expression in different amounts and/or cell types, which may contribute to the targeting of particular self-Ags in memory autoantibody responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

HA104 and HACII mice and the detection of transgenes were described previously (28, 29). Both lineages of HA-transgenic mice were backcrossed to BALB/c mice (Harlan, Indianapolis, IN) at least 10 generations before use in these experiments and were maintained in sterile microisolators at the Wistar Institute Animal Facility. In multiple experiments, nontransgenic littermates of HA104 and HACII mice generated equivalent Ab-secreting cell (ASC) responses as BALB/c mice following virus immunization, and therefore both are referred to as BALB/c mice. C.B-17 scid mice were obtained from the Wistar Institute colony and were screened by the facility to ensure the absence of serum IgM before use in these experiments. All mice were at least 8 wk of age before immunization.

Flow cytometry

BM cells or splenocytes were washed with RPMI 1640 medium (Irvine Scientific, Santa Ana, CA) containing 2% FCS and 0.08% NaN3 and stained with diluted Abs for 30 min. The following Abs were used for analysis: purified anti-CD16/32 (Fc block; clone 2.4G2; BD Pharmingen, San Diego, CA) FITC-anti-mouse CD45R/B220 (clone RA3-6B2; BD Pharmingen), FITC-anti-mouse CD11c (clone HL3; BD Pharmingen), PE-anti-mouse I-Ad (clone AMS-32.1; BD Pharmingen), and biotin-anti-PR8 HA (clone B62-82) (28). Biotin-anti-PR8 HA was detected with streptavidin-Red670 (Invitrogen Life Technologies, Carlsbad, CA). Three-color cytometry was performed on a FACScan flow cytometer (BD Biosciences, San Jose, CA). Data analysis of at least 50,000 events was performed using CellQuest (BD Biosciences). For purification of cells by flow cytometry, samples were sorted to ~95% purity at the Wistar Institute’s sorting facility using a Cytomation MoFlo and Summit software (DakoCytomation, Fort Collins, CO).

Viruses and immunizations

Influenza viruses PR8 (A/Puerto Rico/8/34 (H1N1)), T3 (a mutant PR8 virus containing an aspartic acid to glycine interchange at aa 225) (30), and J1 (a reassortant of PR8 containing the serologically non-cross-reactive H3 subtype HA) (31, 32) were grown in the allantoic cavity of 10-day-old fertilized chicken eggs, purified by sucrose gradient centrifugation, and titered by chicken RBC agglutination (33). To induce primary B cell responses, mice were immunized i.v. (tail vein) with 1000 hemagglutinating units (HAU) T3 virus in 0.2 ml of PBS and sacrificed 5 days later. For secondary B cell responses, mice were primed i.p. with 1000 HAU T3 virus in 0.2 ml of PBS and, after at least 4 wk, they were i.v. boosted with 1000 HAU T3 virus in 0.2 ml of PBS and sacrificed 3 days later.

Virus-specific ELISPOTs

Virus-specific ELISPOTS were done using purified T3, PR8, and J1 viruses as described previously (26). Briefly, 96-well Multiscreen-HA plates (Millipore, Bedford, MA) were coated with 50 µl/well T3, PR8, or J1 virus (800 HAU/ml in PBS plus 0.08% NaN3). After overnight incubation at 4°C, plates were blocked with 0.2 ml/well supplemented with IMDM plus 5% FCS. Splenocytes were plated in duplicate in 0.2 ml at 1 x 106, 2.5 x 105, 6.25 x 104, and 1.25 x 104 cells/well for 4 h at 37°C. Plates were again blocked with 1% BSA in PBS plus 0.08% NaN3. Bound Ab was detected with alkaline phosphatase (AP)-conjugated goat anti-mouse IgM or IgG Ab (Southern Biotechnology Associates, Birmingham, AL). AP was developed with nitroblue tetrazolium (Sigma-Aldrich, St. Louis, MO) and 5-bromo-4-chloro-3-iodolyl phosphate (Sigma-Aldrich) in 0.1 M NaHCO3 plus 0.001 M MgCl2. ASC spots were counted using a dissection microscope. In control experiments, splenocytes from unimmunized mice yielded spots at cell concentrations >106/ml. Accordingly, the lower detection limit for these assays is ~1 ASC/105 splenocytes.

Virus-specific ELISA

ELISAs were done using T3, PR8, and J1 viruses as immunoadsorbents as described previously (26). Briefly, polyvinyl 96-well plates (Costar, Cambridge, MA) were coated with 25 µl of diluted virus (800 HAU/ml in PBS plus 0.08% NaN3), incubated overnight at 4°C, and then blocked with 1% BSA in PBS plus 0.08% NaN3. Hybridoma supernatants were added (diluted in 1% BSA in PBS plus 0.08% NaN3) for 90 min. Bound Ab was detected by using AP-conjugated goat Abs to mouse IgM, total IgG, IgG1, IgG2a, IgG2b, or IgG3 (1 µg/ml in 1% BSA in PBS plus 0.08% NaN3; Southern Biotechnology Associates) and developed using p-nitrophenyl phosphate (Pierce, Rockford, IL). Absorbances were read at 405 nm/650 nm using a microplate reader.

Hybridoma generation

Hybridomas were generated from splenocytes from T3-immunized BALB/c and HACII mice 5 days after primary immunization by fusion with Sp2/0-Ag14 cells and selection with hypoxanthine-azaserine (Sigma-Aldrich) as described previously (26). Ten days after fusion, hybridoma supernatants were aspirated and replaced with IMDM plus 10% FCS. Two days later, supernatants were screened by ELISA for reactivity with T3 and J1 viruses. Those hybridomas that displayed >3-fold higher reactivity with T3 than J1 were expanded in IMDM plus 10% FCS, and supernatants were further screened for reactivity with T3, PR8, and J1 viruses and for IgH chain isotype. Hybridomas were designated as T3 HA specific and PR8 HA specific as previously described (26). In brief, T3 HA-specific hybridoma supernatants displayed ≥10-fold higher reactivity with T3 than PR8 and J1 viruses; PR8 HA-specific hybridoma supernatants displayed equal reactivity with T3 and PR8 viruses and ≥10-fold higher reactivity with T3 and PR8 than with J1 viruses. In the hybridoma designation, the prefix indicates the individual donor mouse and the suffix indicates the individual hybridoma number (e.g., 920-18).

Sequence analysis of Ab V regions

Sequence analysis of hybridoma IgL and IgH chain mRNA V regions was performed as described previously (26). Briefly, mRNA was isolated from ~106 hybridoma cells and reverse transcribed using the {kappa} L chain-specific primer C{kappa}1 and the appropriate H chain constant region-specific primer (cIgM, cIgGx, or cIgG3) under standard conditions (34). PR8 HA-specific B cell clonotypes characteristic of the primary and memory Ab responses of BALB/c mice to PR8 HA, termed C12 and C4, were identified by their use of particular IgH/IgL gene segment combinations. C12 B cells use the V{kappa}C12/VHC12 gene segment combination (35), and C4 B cells use the V{kappa}C4/J{kappa}5 gene segment combination paired with any one of a number of different IgH chain genes (36). PCR was first conducted on the resulting cDNA using C12 and C4 clonotype-specific primers: VHC12 to amplify C12 H chains and V{kappa}C12 and V{kappa}C4 to amplify C12 and C4 L chains, respectively (primers generated by CyberSyn, Lenni, PA). Those cDNA which did not amplify with C12 or C4 clonotype-specific primers were next amplified with the degenerate V{kappa}-specific primer L5 that can amplify a wide variety of V{kappa} gene groups and the degenerate VH-specific primers VH5.1 and VH5.2 that can amplify a wide variety of VH gene groups. Amplified products were run on a 1.5% agarose gel, purified using the Compass DNA purification kit (American Bioanalytical, Natick, MA), and their nucleotide sequences were determined using four-color dye chemistries with the ABI 373S sequencer (PerkinElmer, Wellesley, MA) in the Nucleic Acid Facility at the Wistar Institute.

HA inhibition (HI) assay

The ability of Abs to inhibit viral agglutination of chicken RBC was measured as described previously (33). Briefly, 4 HAU of T3 or PR8 virus (25 µl PBS) were plated in 96-well V-bottom Multiscreen-HA plates (Nunc, Roskilde, Denmark). To prepare serum, blood clotted at room temperature for 45 min was centrifuged, and serum was removed and diluted 1/10 in 1% BSA in PBS plus 0.08% NaN3. Two- or 3-fold dilutions of serum (25 µl of PBS), beginning with 1/50, were added to the virus and incubated for 1 h at room temperature. Fifty microliters of 1% chicken RBC was added and incubated for 30 min. HA inhibition (HI) titers were determined similarly for purified Abs. HI titers were determined as the highest serum dilution or the lowest Ab concentration at which HA of chicken RBC was inhibited, for serum and purified Ab samples, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PR8 HA is an abundant membrane-bound self-Ag in HACII mice

HACII mice were previously generated by linking DNA encoding the PR8 HA to sequences encoding a portion of the MHC class II I-E{alpha} promoter (28). HA transgene mRNA was detected in a variety of tissues in HACII mice, including BM and spleen, as had previously been described for HA104 mice (data not shown) (26). We also used flow cytometry to examine BM cells and splenocytes from HACII, HA104, and BALB/c mice for their expression of cell surface HA. HA expression was found to increase in parallel with MHC class II on BM cells from HACII mice, but was not detected on BM cells obtained either from BALB/c or HA104 mice (Fig. 1A). Indeed, more than one-half of the B220+ BM cells from HACII mice expressed high levels of cell surface PR8 HA (Fig. 1B). PR8 HA could also be detected on a fraction of splenocytes from HACII (but not BALB/c or HA104) mice when analyzed directly ex vivo; these PR8 HA+ cells appeared to be predominantly immature B cells based on their low levels of MHC class II and IgD (Fig. 1, A and B, and data not shown). When stimulated in vitro with anti-IgM or with IL-4 and a CD154 fusion protein, the majority of B220+ splenocytes from HACII mice (but not from BALB/c or HA104 mice) expressed cell surface HA (Fig. 1C and data not shown). Splenic CD11c+ cells from HACII mice also expressed low levels of cell surface HA when analyzed directly ex vivo, but following overnight culture in vitro all of the CD11c+ cells from HACII mice (but not those from BALB/c or HA104 mice) expressed cell surface HA (Fig. 1C). Together, these data indicate that PR8 HA is expressed as a cell surface Ag that is abundantly available for recognition by developing B220+ cells in the BM of HACII mice. PR8 HA is also constitutively expressed by MHC class II+ splenocytes of HACII mice, and its expression can increase substantially following receipt of signals that stimulate MHC class II up-regulation. By contrast, PR8 HA expression was not detectable in HA104 mice by flow cytometry, even though its presence induces negative selection of a population of PR8 HA-specific B cells from the primary B cell repertoire (see below) (26).



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  		FIGURE 1. PR8 HA is a membrane-bound self-Ag in HACII mice. A, Dot plots show the levels of MHCII vs PR8 HA expression on BM and splenic lymphocytes isolated from BALB/c, HACII, and HA104 mice. B, Histograms show the levels of PR8 HA expression on BM and splenic B220+ cells isolated from BALB/c, HACII, and HA104 mice. C, Histograms show the levels of PR8 HA expression on BALB/c, HACII, and HA104 B cells (B220+) and dendritic cells (CD11c+) directly ex vivo (upper panels) and following in vitro stimulation (lower panels).

 
Use of mutant virus immunization to examine B cell tolerance in HACII and HA104 mice

T3 is a mAb-selected influenza virus that is identical to PR8 except for a single Asp to Gly interchange at aa 225 (Fig. 2A and Ref. 30). This mutation is located in one of the four major B cell epitopes on the HA and does not alter any of the eight known CD4+ T cell determinants recognized in BALB/c mice (30, 37). We previously showed that the majority of the Ab response to the T3 HA in BALB/c mice cross-reacts with the PR8 HA, while ~15% is directed to epitopes that include aa 225, uses structurally distinct variable regions, and does not cross-react with the PR8 HA (38). To examine the effects of the neo self-PR8 HA on B cell repertoire formation, we immunized HACII, HA104, and BALB/c mice with T3 virus and determined the frequency of B cells that can react with T3 and PR8 viruses. We also determined the frequency that could react with the reassortant virus J1, which contains all of the non-HA proteins of PR8 virus but a serologically non-cross-reactive (H3 vs H1) subtype HA (Fig. 2A and Refs. 31 and 32). This allowed the frequency of B cells directed to non-HA viral components (i.e., other virus proteins and components such as carbohydrates that derive from propagating the virus in hen’s eggs) to be quantitated. As shown schematically in Fig. 2B, the number of B cells that could react with T3, but not with PR8, quantitated the T3 HA-specific B cells that recognize the mutated B cell epitope present in the T3 HA. Likewise, the number that could react with both T3 and PR8, but not with J1, quantitated PR8 HA-specific B cells; these recognize epitopes shared between the T3 and PR8 HA molecules and that are self-epitopes in HACII and HA104 mice. Immunization with T3 virus therefore allowed the development of autoreactive PR8 HA-specific B cells in HA-transgenic mice to be quantitated relative to those directed to non-self-epitopes on the same molecule. In addition, using whole T3 virus as the immunogen ensured that CD4+ T cells directed to abundant non-HA viral proteins could provide a source of intermolecular cognate help for HA-specific B cells (20, 39).



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  		FIGURE 2. Fine specificity of the B cell response to T3 virus. A, Schematic diagram representing the HA, neuraminidase, M2, and carbohydrate components of T3, PR8, and J1 viruses. The subtypes of the viruses (e.g., H1N1) are indicated. B, Schematic representation showing the use of T3 (T), PR8 (P), and J1 (J) viruses to measure the fine specificity of the B cell response to the mutant epitope of T3 HA (non-self- HA in HA-transgenic mice), PR8 HA (self-HA in HA-transgenic mice), and non-HA components of T3 virus as measured by ELISPOT.

 
HA negatively selects primary response IgG-secreting B cells more efficiently than IgM-secreting B cells

We first used ELISPOT analysis to determine the frequency of ASC that were induced 5 days following primary immunization with T3 virus. Splenocytes from BALB/c mice contained HA-specific IgG and IgM ASC; the IgG ASC were approximately twice as abundant as IgM ASC and were predominantly PR8 HA specific (average frequency, 29.9 ASC/105 splenocytes; Fig. 3, A, D, and G, and Table I). PR8 HA-specific IgG ASC were barely detectable in HACII mice (≤1 ASC/105 splenocytes; Fig. 3H and Table I) and, as previously described, the average frequency of PR8 HA-specific IgG ASC was also substantially reduced in HA104 mice (4.9 ASC/105 splenocytes; Fig. 3I and Table I) (26). Significantly, however, both HACII and HA104 mice retained the ability to generate IgG ASC that could react with the non-self-epitope of the T3 HA, since the average frequencies of IgG ASC that reacted with T3 but not with PR8 were similar in BALB/c, HACII, and HA104 mice (15.2, 9.2, and 11.0 ASC/105 splenocytes, respectively; Fig. 3, D–F, and Table I). Thus, PR8 HA-specific IgG ASC that dominate the primary B cell response to T3 virus in BALB/c mice are negatively selected in HACII mice because of their specificity for the neo-self-PR8 HA, as previously described in HA104 mice (26).



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  		FIGURE 3. Fine specificity of HA-specific primary responses in T3-immunized BALB/c, HACII, and HA104 mice. A–C, Bar graphs show the average numbers of T3 (T), PR8 (P), and J1 (J) virus-specific IgM and IgG ASC measured in splenocytes from BALB/c (n = 12), HACII (n = 12), and HA104 mice (n = 10) by ELISPOT 5 days after primary immunization with T3 virus. The average numbers of T3 HA-specific (D–F) and PR8 HA-specific (G–I) IgM and IgG splenic ASC are also shown. Each dot represents an individual mouse.

 

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  		Table I. Fine specificity of the primary Ab response of BALB/c, HACII, and HA104 mice

 
The frequencies of PR8 HA-specific IgM ASC were much more similar in the different lineages. Strikingly, the average frequency of PR8 HA-specific IgM ASC in HACII mice (7.0 ASC/105 splenocytes) was reduced by only one-half relative to BALB/c mice, sharply contrasting the ~30-fold reduction in the relative frequencies of PR8 HA-specific IgG ASC (Fig. 3E and Table I). Similarly, PR8 HA-specific IgM ASC were equally abundant in T3-immunized HA104 and BALB/c mice (average frequencies, 14.0 vs 15.2 ASC/105 splenocytes, respectively; Fig. 3, D and F, and Table I), as previously described (26). Thus, even in HACII mice where the PR8 HA is an abundantly expressed membrane bound self-Ag, it imposed only modest reductions in the frequencies of PR8 HA-specific IgM ASC that could be induced by T3 immunization.

To further characterize the primary Ab response of HACII mice, we generated hybridoma panels from BALB/c and HACII mice 5 days following primary immunization with T3 virus. PR8 HA-specific IgG-secreting hybridomas were isolated in far lower numbers from HACII than BALB/c mice; 75 PR8 HA-specific IgG-secreting hybridomas were obtained from BALB/c mice and only four were obtained from HACII mice, while T3 HA-specific IgG-secreting hybridomas were isolated at similar frequencies (Fig. 4A). PR8 HA-specific IgM-secreting hybridomas were also isolated at comparable frequencies from HACII and BALB/c mice (Fig. 4A), despite the pronounced reductions in the frequencies of PR8 HA-specific IgG-secreting hybridomas obtained from HACII mice. These hybridoma analyses therefore closely correlated with the ELISPOT data indicating that PR8 HA-specific IgM-secreting B cells are less susceptible to negative selection than IgG-secreting B cells in HACII mice.



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  		FIGURE 4. Specificity and clonotype usage of HA-specific hybridomas generated from T3-immunized HACII mice. A, The total number of hybridomas and of T3 HA-specific and PR8 HA-specific hybridomas generated from BALB/c (n = 3) and HACII (n = 3) splenocytes 5 days after primary immunization with T3 virus. B, VH and V{kappa} gene families, as well as the JH and J{kappa} gene segments used by PR8 HA-specific hybridomas generated from individual HACII mice (3511, 3513, and 756) were determined by nucleotide sequences assigned to previously described families (57 58 ). C12 B cells were identified by the use of the prototypic V{kappa}C12/VHC12 gene segment combination. No B cell hybridomas bearing the C12 clonotype were identified. Usage of the V{kappa}C4/J{kappa}5 gene segment combination that is characteristic of the C4 clonotype is shown in bold; the nucleotide sequences of these hybridomas were all identical to prototypic V{kappa}C4/J{kappa}5 L chains. Hybridomas that yielded mixed V{kappa} gene sequences using the degenerate V{kappa} gene primer, but that did not amplify with the V{kappa}C12- or V{kappa}C4-specific primers are indicated as "Not C4/C12." The nucleotide sequences of these V regions are available from European Molecular Biology Laboratory/GenBank/DNA Data Bank of Japan under accession numbers AY603667–AY603683.

 
A clonotype characteristic of memory responses is induced by primary immunization of HACII mice

The B cell response of BALB/c mice to PR8 HA is comprised of two functionally distinct B cell subsets that can be identified based on the sequences of their Ig variable regions. One subset contributes to the IgG component of the primary Ab response of BALB/c mice to PR8 virus, but fails to form B cell memory. These C12 B cells use the V{kappa}C12 L chain paired with one of two IgH chain genes, VHC12.1 or VHC12.2 (35, 40). The second subset participates in primary responses as primarily IgM Ab-secreting cells, but also undergoes somatic mutation and affinity maturation of their Ag receptors to develop into memory B cells. These C4 B cells use the V{kappa}C4/J{kappa}5 gene segment combination paired with a number of different VH gene family genes (36). We examined the PR8 HA-specific hybridomas obtained from HACII mice for the usage of C12 and C4 variable region clonotypes that are characteristic of distinct phases of the immune response to PR8 HA in BALB/c mice.

Approximately one-half of the PR8 HA-specific IgG-secreting B cell hybridomas induced by primary immunization of BALB/c mice use the C12 clonotype, although C12 B cells do not participate in memory responses to PR8 HA (35, 40). None of the PR8 HA-specific hybridomas generated from HACII mice used the C12 clonotype (Fig. 4B), and we previously showed that C12 B cells are likewise greatly underrepresented among PR8 HA-specific hybridomas generated from T3-immunized HA104 mice (26). C4 B cells, in contrast, are typically isolated as unmutated IgM-secreting hybridomas following primary immunization of BALB/c mice and, unlike C12 B cells, C4 B cells can also be isolated as somatically mutated IgG-secreting hybridomas following secondary immunization (36). We previously showed that one-half of the IgM-secreting hybridomas obtained from HA104 mice used the C4 clonotype (26) and subsequently showed that somatically mutated C4 B cells could be isolated from memory responses of HA104 mice (in which PR8 HA-specific memory responses are unaffected by negative selection; see below) (27). When we analyzed the PR8 HA-specific IgM-secreting hybridomas from HACII mice, we again found that one-half use the C4 clonotype (Fig. 4B). These hybridoma analyses support the conclusion based on ELISPOT analysis that PR8 HA-specific primary response IgG-secreting B cells (including C12) undergo substantial negative selection in HACII mice because of their specificity for the neo self-HA, as previously described in HA104 mice (26). By contrast, the frequency of PR8 HA-specific IgM-secreting B cells (including C4) that can be induced by virus immunization is much less affected by negative selection even in HACII mice, in which the PR8 HA is a membrane-bound Ag that is abundantly available for recognition by developing B cells.

The IgM Ab responses to PR8 HA in HACII, HA014, and BALB/c mice are qualitatively similar

We examined sera from HACII, HA104, and BALB/c mice obtained 5 days following primary T3 immunization for their reactivity with the PR8 HA in HI assays, which require that Abs recognize conformation-dependent epitopes on the HA with sufficient avidity to inhibit virus attachment (33). The HI titers of HACII mice were reduced by only one-half, on average, relative to those from BALB/c mice, roughly corresponding to the reduced frequencies of PR8 HA-specific ASC measured by ELISPOT (Fig. 5A). These data suggest that the PR8 HA-specific IgM B cells that were detected in HACII mice are not of unusually low affinity relative to those from BALB/c mice, nor directed toward denatured epitopes that might be present in the virus preparations.



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  		FIGURE 5. HA inhibitory activity and relative affinity of PR8 HA-specific Abs from BALB/c, HACII, and HA104 mice. A, Bar graphs show the average highest dilution (HI titer) at which serum from BALB/c (n = 11), HACII (n = 9), and HA104 (n = 12) mice obtained 5 days after primary immunization with T3 virus-inhibited RBC agglutination by PR8 virus in a HI assay. The dashed line indicates values obtained from nonimmunized BALB/c serum. Each dot represents serum prepared from an individual mouse. B, The lowest Ab concentration at which individual purified C4 Abs from BALB/c (hybridomas 963-144, 963-184, 922-1418), HACII (hybridomas 3511-49, 3513-96, 756-681), and HA104 (hybridomas 920-177, 921-143, 921-182) mice inhibited RBC agglutination in a HI assay. Background in the HI assay was 1100 µg/ml, as determined by measuring the ability of a purified BALB/c IgM Ab (MOPC 104E, Sigma catalogue no. M-5909) to inhibit RBC agglutination by PR8 virus. C–E, Line graphs show the binding of purified C4 IgM Abs from BALB/c (hybridomas 963-144, 963-184, 922-1418), HACII (hybridomas 3511-49, 3513-96, 756-681), and HA104 mice (hybridomas 920-177, 921-143, 921-182) (•) to PR8 virus in an ELISA. All panels also show binding by the purified C12 IgM Ab 920-108 ({square}). F, Line graphs show the binding of purified C12 IgG Abs from BALB/c (hybridomas 963-100, 922-64, 922-72, 922-122, 963-206, 922-177) to PR8 virus in an ELISA. Binding by the purified C12 IgG Ab 920-18 is also shown ({triangleup}). Each line represents Ab purified from an individual hybridoma. Hybridomas obtained from BALB/c and HA104 mice were previously described (26 ).

 
We also purified Abs from C4 IgM-secreting hybridomas obtained from HACII, HA104, and BALB/c mice and examined their reactivity with the PR8 HA. These were comparable whether measured by HI (Fig. 5B) or ELISA (Fig. 5, C–E), providing further evidence that the PR8 HA-specific IgM Abs from HACII mice do not possess unusually low affinities for the PR8 HA. In addition, we were interested in comparing the reactivity of the C4 Abs with those of the C12 Abs, which undergo more efficient negative selection in HACII and HA104 mice. Because C12 IgG and C4 IgM Abs have different valencies that affect their avidity, we could not compare their reactivity directly. We could however assess their reactivity relative to a pair of Abs that use identical C12 variable region genes in conjunction with either IgG2a (920-18) or IgM (920-108) constant regions that we previously obtained from a T3-immunized HA104 mouse (26). The six C12 Abs (all of IgG2a isotype) from BALB/c mice exhibited reactivity with the PR8 HA equal to or up to 100-fold greater than the 920-18 Ab (Fig. 5F). By contrast, the C4 IgM Abs from BALB/c, HA104 and HACII mice all exhibited reactivities toward the PR8 HA that were 10- to 100-fold lower than those exhibited by the 920-108 Ab (Fig. 5, C–E). These data suggest that C12 IgG Abs, which are subject to more efficient negative selection from the primary B cell repertoires of HACII and HA104 mice, may also typically possess higher intrinsic affinities for the PR8 HA than C4 IgM Abs.

PR8 HA-specific B cells are negatively selected during memory repertoire formation in HACII mice

To determine whether PR8 HA-specific IgM-secreting B cells that participate in primary responses might be subject to negative selection during memory formation, we analyzed the memory B cell responses of BALB/c, HACII, and HA104 mice to reimmunization with T3 virus. BALB/c splenocytes contained a substantial number of PR8 HA-specific IgG ASC 3 days following secondary immunization (average frequency, 67.6 ASC/105 splenocytes; Fig. 6G and Table II). As was observed in the primary response, a small component of the HA-specific IgG ASC response reacted only with T3 virus (average frequency, 11.2 ASC/105 splenocytes; Fig. 6D and Table II). As previously described, the magnitude and specificity of the responses in T3-immunized HA104 mice were comparable to those induced in BALB/c mice (57.7 PR8 HA-specific IgG ASC/105 splenocytes and 13.0 T3 HA-specific IgG ASC/105 splenocytes; Fig. 6, F and I, and Table II) (27). In sharp contrast, PR8 HA-specific ASC were absent from HACII splenocytes following secondary challenge with T3 virus (Fig. 6H and Table II). Remarkably, the average numbers of HA-specific IgG ASC that were induced in T3-immunized BALB/c, HA104, and HACII mice were equivalent (78.8, 70.7, and 82.1 ASC/105 splenocytes, respectively; Fig. 6, A–C). However, in HACII mice, these HA-specific IgG ASC were entirely specific for the non-self-epitopes on the T3 HA; HACII mice contained on average 82.1 T3 HA-specific IgG ASC/105 splenocytes, whereas BALB/c and HA104 mice contained on average 11.2 and 13.0 T3 HA-specific IgG ASC/105 splenocytes, respectively (Fig. 6, B, E, and H, and Table II). The absence of PR8 HA-specific IgG ASC in HACII mice was therefore accompanied by a corresponding >5-fold increase in the frequency directed to the non-self-T3 epitopes and equals the total number of HA-specific IgG ASC generated in BALB/c mice. The specificity of the serum Ab response was also entirely focused toward T3 epitopes and was of equal magnitude to the entire HA-specific Ab response of BALB/c mice (Fig. 6J). Thus, in HACII mice, negative selection of PR8 HA-specific B cells focused the specificity of the memory B cell response toward non-self-T3 epitopes and away from reactivity with the PR8 HA. The total magnitude of the HA-specific memory response that was generated nevertheless remained constant, because increased formation of memory B cells directed to non-self-T3 epitopes precisely compensated for the absence of PR8 HA-specific B cells.



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  		FIGURE 6. Fine specificity of HA-specific secondary responses in T3-immunized BALB/c, HACII, and HA104 mice. A–C, Bar graphs show the average number of T3 (T), PR8 (P), and J1 (J) virus-specific IgM and IgG ASC measured in splenocytes from BALB/c (n = 8), HACII (n = 7), and HA104 mice (n = 4) by ELISPOT 3 days after secondary immunization with T3 virus. The average numbers of T3 HA-specific (D–F) and PR8 HA-specific (G–I) IgM and IgG splenic ASC are also shown. Each dot represents an individual mouse. J, Bar graphs show the average highest dilution (HI titer) at which serum from BALB/c (n = 8), HACII (n = 7),and HA104 (n = 6) mice obtained 3 days after secondary immunization with T3 virus-inhibited RBC agglutination by T3 (T) and PR8 (P) viruses in a HI assay. Serum obtained from nonimmunized BALB/c mice yielded titers of 1/50 in these assays. Each dot represents serum prepared from an individual mouse.

 

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  		Table II. Fine specificity of the secondary Ab response of BALB/c, HACII, and HA104 mice

 
B cells from HACII mice mediate dominant negative selection during PR8 HA-specific memory B cell formation

To examine whether the PR8 HA expressed as a self-Ag in HACII mice could mediate dominant negative selection of B cells that had developed in its absence, C.B-17 scid mice were reconstituted with splenocytes from BALB/c or HACII mice, or an equal mixture of each, and analyzed for their memory responses to T3 virus. Mice reconstituted with splenocytes from either BALB/c or HACII mice generated HA-specific IgG ASC with specificities for T3 and PR8 that closely matched those of the intact BALB/c or HACII mice (Fig. 7A). Those reconstituted with a mixture of BALB/c and HACII splenocytes generated HA-specific Ab responses that were entirely specific for T3 virus (Fig. 7A), and purified B220+ cells from HACII mice induced a similar specificity shift when mixed with BALB/c splenocytes (Fig. 7B). Thus, B220+ cells from HACII mice mediated negative selection of PR8 HA-specific B cells during memory B cell repertoire formation, even though those B cells had developed in the absence of the PR8 HA. Moreover, in this case also, negative selection of PR8 HA-specific memory B cells was accompanied by a compensatory increase in the formation of memory B cells directed to non-self-T3 epitopes.



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  		FIGURE 7. B cells from HACII mice induce dominant negative selection of PR8 HA-specific B cells. A, C.B-17 scid mice were seeded with either no cells, 3.0 x 107 BALB/c splenocytes, 3.0 x 107 HACII splenocytes, or 3.0 x 107 of a 50:50 mixture of BALB/c and HACII splenocytes, primed 1 day later with T3 virus, and then boosted 1 mo later with T3 virus. Bar graphs show the average number of T3 (T), PR8 (P), and J1 (J) virus- specific IgG ASC measured in recipient spleens by ELISPOT 3 days after secondary immunization (n = 3). B, C.B-17 scid mice were seeded with a mixture of 1.5 x 107 BALB/c splenocytes and either 1.0 x 107 FACS-purified B220+ splenic B cells from BALB/c or HACII mice, as indicated. Bar graphs show the average number of T3 (T), PR8 (P), and J1 (J) virus-specific IgG ASC measured in recipient spleens by ELISPOT 3 days after secondary immunization (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have examined how self-tolerance affects the specificity, magnitude, and composition of B cell responses in HACII mice, in which PR8 HA is a membrane-bound self-Ag that is abundantly available for recognition by developing B cells. Using mice containing unmanipulated Ig repertoires and T3 mutant virus immunization, we showed that IgG-secreting B cells (including C12) that dominate primary responses to PR8 HA in BALB/c mice were profoundly reduced in HACII mice, while IgM-secreting B cells (including C4) were much less sensitive to negative selection from the primary repertoire. The PR8 HA-specific IgM-secreting B cells failed however to enter the memory repertoire of HACII mice, and there was a net increase in the production of memory B cells that could recognize the non-self-epitopes on the T3 HA molecule. These studies provide direct evidence that autoreactive B cells can be excluded from memory formation based on their specificity for a membrane-bound self-Ag. They also suggest that selection processes occurring during memory formation act to fill an available niche, because the formation of T3-specific memory B cells increased in HACII mice to precisely compensate for the absence of PR8 HA-specific B cells.

The finding that PR8 HA-specific B cells from BALB/c mice were eliminated from memory responses by mixing with splenocytes or B cells from HACII mice indicated that negative selection is a dominant process. Since these mice also retained the ability to generate T3-specific memory B cell responses, it is unlikely that a lack of CD4+ T cell help or deletion by complement-mediated cytotoxicity or Ab-dependent cellular cytotoxicity caused PR8 HA-specific B cells to be eliminated during memory formation. Rather, signals transmitted to PR8 HA-specific B cells by the neo self-HA via the B cell receptor instructed these B cells to fail to enter into the memory B cell compartment. PR8 HA-specific B cells may be prevented from memory formation in HACII mice because they are rendered anergic; for example, anergic B cells may be able to develop into primary response ASC, but be incapable of progression into memory cells. Since we found that PR8 HA-specific B cells from BALB/c mice were equally sensitive to elimination from memory responses when mixed with B cells from HACII mice, this too could reflect the induction of anergy induced by exposure to PR8 HA-expressing B cells from HACII mice, although in this case it would be acting on mature B cells that had not been anergized during their development in the BM (41). If this is the case, the induction of anergy could provide a mechanistic basis for establishing tolerance in the memory compartment by limiting the functional potential of cells that evade negative selection from the primary repertoire.

It is also possible that processes acting in the germinal center pathway lead to the negative selection of PR8 HA-specific B cells during memory formation in HACII mice. High doses of soluble Ags have been shown to induce apoptosis of germinal center B cells (22, 23), and introduction of a Bcl-2 transgene allowed B cells with dual specificity for a foreign and self-Ag to be recovered from germinal center B cells (24, 25). If B cells develop an increased sensitivity to apoptosis as they enter the germinal center pathway (as suggested by elevated levels of Fas expression) (42, 43, 44), the ubiquitous expression of PR8 HA could induce apoptosis of PR8 HA-specific B cells more rapidly than T3 HA-specific B cells and T3 HA-specific B cells may then be at a competitive advantage for niches that can be filled by cells receiving positively selecting or survival signals from CD4+ T cells. The defining characteristic of somatic hypermutation in B cells is the reproducible selection of rare cells that have enhanced reactivity toward an immunizing agent, implying that their selection is based on competition for niches that provide survival signals based on goodness of fit (2, 3, 4). The studies here show that competition could also provide the basis for elimination of autoreactive B cells, since the number of T3 HA-specific B cells that were selected into the memory B cell repertoire in HACII mice increased substantially relative to BALB/c mice at the expense of those directed to the self-PR8 HA.

Our previous studies in HA104 mice provided evidence that separate populations of PR8 HA-specific B cells (i.e., C12 vs C4) differ in their sensitivity to negative selection from the primary B cell repertoire, although the possibility existed that C4 B cells might evade negative selection in HA104 mice because the HA is expressed at low levels or by cell types that are poorly accessible to developing B cells rather than differences in negative selection potential between C12 and C4 B cells (45, 46). In HACII mice, however, the PR8 HA is expressed as a membrane-bound Ag by a large fraction of B220+ cells in the BM; therefore, it is unlikely that C4 B cells were not exposed to the PR8 HA during their development. Instead, intrinsic differences between C12 and C4 B cells appear to affect their susceptibility to negative selection from the primary B cell repertoire. What could account for their differing sensitivities? C12 B cells appear on average to have higher relative affinities for the PR8 HA than C4 B cells, suggesting that affinity for the neo self-HA could contribute to the sensitivity of PR8 HA-specific B cells to negative selection in HA104 and HACII mice. However, in models where deletion and receptor editing of autoreactive B cells has been shown to occur, autoreactive B cells with a wide range of affinities for self-Ags were shown to undergo tolerance induction (47, 48). Also, C4 IgM Abs and indeed serum Ab responses generated following primary immunization appeared to be of comparable affinity for the PR8 HA in HACII, HA104, and BALB/c mice, arguing that low affinity is not the sole reason that these cells evade negative selection.

A second possibility is that distribution among different cell subsets affects the susceptibility of C12 (IgG-secreting) vs C4 (IgM-secreting) B cells to negative selection from the primary repertoire. For example, C12 B cells might derive from marginal zone B cells, which undergo rapid terminal differentiation into ASC to provide a burst of Ab production early during primary immune responses to blood-borne particulate Ags (49, 50, 51, 52). These properties are consistent with the abrupt expansion, class switching, and terminal differentiation of C12 B cells in response to influenza virus PR8 HA (40). The higher relative affinities of C12 B cells for the PR8 HA could then in part reflect their activation from a compartment in BALB/c mice (e.g., marginal zone B cells) that might favor high-affinity B cells (53), while processes that direct development of this compartment might also enhance sensitivity to negative selection. It also remains possible that interactions with the neo self-HA could have altered the development of PR8 HA-specific B cells in HACII mice, such that the PR8 HA-specific IgM responses induced by primary virus immunization derive from distinct B cell subsets in HACII vs BALB/c mice (54). However, PR8 HA-specific B cells from BALB/c mice were also subjected to negative selection by HACII splenocytes, indicating that the failure of C4 IgM-secreting B cells to mature into memory B cells cannot be solely explained by their activation from a different B cell subset. Future studies using transgenic mice expressing C12 and C4 Ig molecules should help clarify factors governing the differing fates of these populations. Our findings at this stage nevertheless complement previous studies in which transgenic mice expressing autoreactive Igs were shown in some models to be subject to overt deletion (11, 12), whereas in other systems they could persist in the primary repertoire (15, 16, 55, 56). We have shown that separate populations of autoreactive B cells can differ in their ability to be prevented from development and activation from the primary repertoire, even when directed to an abundant membrane-bound self-Ag.

Finally, it is worth noting that the negative selection of HA-specific B cells, when it occurred, was highly specific; HACII and HA104 mice each retained the ability to mount primary IgG Ab responses to the non-self-epitopes on the T3 HA molecule, with similar frequencies to BALB/c mice. Likewise, negative selection of PR8 HA-specific B cells during memory formation in HACII mice led to a complete skewing of the Ab response toward reactivity with non-self-epitopes on the T3 HA. By contrast, the memory responses of HA104 mice to T3 virus were as cross-reactive with the PR8 HA as those of BALB/c mice. Although somatically mutated autoantibodies are a hallmark of certain autoimmune diseases (e.g., systemic lupus erythematosus) (5, 6, 7), factors that allow or promote their development remain poorly defined. Because PR8 HA-specific memory B cell formation is unaffected in HA104 mice, the studies here provide evidence that only a subset of self-Ags (such as those expressed at high densities as membrane-bound Ags) may be able to induce negative selection of autoreactive memory B cells. It will be important in future studies to more fully elucidate how expression in different amounts or cell types contributes to the development and targeting of autoantibody responses to particular self-Ags.


   Acknowledgments
 
We thank Andrew Rankin for his thoughtful discussions, Andrea Holenbeck, Christina Young, Cristina Cozzo, and Dr. Joseph Larkin III for their assistance, and Dr. Jan Erikson and Dr. Michael Cancro for critical reading of this manuscript.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by grants from the National Institutes of Health, by the Lupus Foundation of Southeastern Pennsylvania, and by the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health. Back

2 Address correspondence and reprint requests to Dr. Andrew J. Caton, Wistar Institute, Room 262, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: caton{at}wistar.upenn.edu Back

3 Abbreviations used in this paper: BM, bone marrow; HA, hemagglutinin; PR8, influenza virus A/PR/8/34; ASC, Ab-secreting cell; HAU, HA unit; HI, HA inhibition; AP, alkaline phosphatase. Back

Received for publication June 25, 2004. Accepted for publication August 20, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Alt, F. W., T. K. Blackwell, G. D. Yancopoulos. 1987. Development of the primary antibody repertoire. Science 238:1079.[Abstract/Free Full Text]
  2. Przylepa, J., C. Himes, G. Kelsoe. 1998. Lymphocyte development and selection in germinal centers. Curr. Top. Microbiol. Immunol. 229:85.[Medline]
  3. Manser, T., K. M. Tumas-Brundage, L. P. Casson, A. M. Giusti, S. Hande, E. Notidis, K. A. Vora. 1998. The roles of antibody variable region hypermutation and selection in the development of the memory B-cell compartment. Immunol. Rev. 162:183.[Medline]
  4. Tarlinton, D. M., A. Light, G. J. Nossal, K. G. Smith. 1998. Affinity maturation of the primary response by V gene diversification. Curr. Top. Microbiol. Immunol. 229:71.[Medline]
  5. Radic, M. Z., M. Weigert. 1994. Genetic and structural evidence for antigen selection of anti-DNA antibodies. Annu. Rev. Immunol. 12:487.[Medline]
  6. Shlomchik, M. J., A. Marshak-Rothstein, C. B. Wolfowicz, T. L. Rothstein, M. G. Weigert. 1987. The role of clonal selection and somatic mutation in autoimmunity. Nature 328:805.[Medline]
  7. Diamond, B., J. B. Katz, E. Paul, C. Aranow, D. Lustgarten, M. D. Scharff. 1992. The role of somatic mutation in the pathogenic anti-DNA response. Annu. Rev. Immunol. 10:731.[Medline]
  8. Reichlin, M.. 1972. Localizing antigenic determinants in human haemoglobin with mutants: molecular correlations of immunological tolerance. J. Mol. Biol. 64:485.[Medline]
  9. Hammerling, G. J., J. W. Chamberlain, O. Dill, S. Y. Yang, B. Dupont, R. Khan, R. Chua, S. M. Weissman, U. Hammerling. 1990. Self-tolerance to HLA focuses the response of immunized HLA-transgenic mice on production of antibody to precise polymorphic HLA alloantigens. Proc. Natl. Acad. Sci. USA 87:235.[Abstract/Free Full Text]
  10. Minnerath, J. M., C. M. Mueller, S. Buron, R. Jemmerson. 1995. B lymphocyte recognition of cytochrome c: higher frequency of cells specific for self versus foreign antigen early in the immune response and V gene usage in the response to self antigen. Eur. J. Immunol. 25:784.[Medline]
  11. Nemazee, D. A., K. Burki. 1989. Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature 337:562.[Medline]
  12. Chen, C., Z. Nagy, M. Z. Radic, R. R. Hardy, D. Huszar, S. A. Camper, M. Weigert. 1995. The site and stage of anti-DNA B-cell deletion. Nature 373:252.[Medline]
  13. Tiegs, S. L., D. M. Russell, D. Nemazee. 1993. Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 177:1009.[Abstract/Free Full Text]
  14. Gay, D., T. Saunders, S. Camper, M. Weigert. 1993. Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177:999.[Abstract/Free Full Text]
  15. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676.[Medline]
  16. Erikson, J., M. Z. Radic, S. A. Camper, R. R. Hardy, C. Carmack, M. Weigert. 1991. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature 349:331.[Medline]
  17. Mandik-Nayak, L., A. Bui, H. Noorchashm, A. Eaton, J. Erikson. 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.[Abstract/Free Full Text]
  18. Cyster, J. G., S. B. Hartley, C. C. Goodnow. 1994. Competition for follicular niches excludes self-reactive cells from the recirculating B-cell repertoire. Nature 371:389.[Medline]
  19. Steinhoff, U., C. Burkhart, H. Arnheiter, H. Hengartner, R. Zinkernagel. 1994. Virus or a hapten-carrier complex can activate autoreactive B cells by providing linked T help. Eur. J. Immunol. 24:773.[Medline]
  20. Zinkernagel, R. M., S. Cooper, J. Chambers, R. A. Lazzarini, H. Hengartner, H. Arnheiter. 1990. Virus-induced autoantibody response to a transgenic viral antigen. Nature 345:68.[Medline]
  21. Seo, S. J., M. L. Fields, J. L. Buckler, A. J. Reed, L. Mandik-Nayak, S. A. Nish, R. J. Noelle, L. A. Turka, F. D. Finkelman, A. J. Caton, J. Erikson. 2002. The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity 16:535.[Medline]
  22. Pulendran, B., G. Kannourakis, S. Nouri, K. G. Smith, G. J. Nossal. 1995. Soluble antigen can cause enhanced apoptosis of germinal-centre B cells. Nature 375:331.[Medline]
  23. Shokat, K. M., C. C. Goodnow. 1995. Antigen-induced B-cell death and elimination during germinal-centre immune responses. Nature 375:334.[Medline]
  24. Notidis, E., L. Heltemes, T. Manser. 2002. Dominant, hierarchical induction of peripheral tolerance during foreign antigen-driven B cell development. Immunity 17:317.[Medline]
  25. Hande, S., E. Notidis, T. Manser. 1998. Bcl-2 obstructs negative selection of autoreactive, hypermutated antibody V regions during memory B cell development. Immunity 8:189.[Medline]
  26. Caton, A. J., J. R. Swartzentruber, A. L. Kuhl, S. R. Carding, S. E. Stark. 1996. Activation and negative selection of functionally distinct subsets of antibody-secreting cells by influenza hemagglutinin as a viral and a neo-self antigen. J. Exp. Med. 183:13.[Abstract/Free Full Text]
  27. Reed, A. J., M. P. Riley, A. J. Caton. 2000. Virus-induced maturation and activation of autoreactive memory B cells. J. Exp. Med. 192:1763.[Abstract/Free Full Text]
  28. Reed, A. J., H. Noorchashm, S. Y. Rostami, Y. Zarrabi, A. R. Perate, A. N. Jeganathan, A. J. Caton, A. Naji. 2003. Alloreactive CD4 T cell activation in vivo: an autonomous function of the indirect pathway of alloantigen presentation. J. Immunol. 171:6502.[Abstract/Free Full Text]
  29. Cerasoli, D. M., J. McGrath, S. R. Carding, F. F. Shih, B. B. Knowles, A. J. Caton. 1995. Low avidity recognition of a class II-restricted neo-self peptide by virus-specific T cells. Int. Immunol. 7:935.[Abstract/Free Full Text]
  30. Caton, A. J., G. G. Brownlee, J. W. Yewdell, W. Gerhard. 1982. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell 31:417.[Medline]
  31. Palese, P.. 1977. The genes of influenza virus. Cell 10:1.[Medline]
  32. Palese, P., J. L. Schulman. 1976. Mapping of the influenza virus genome: identification of the hemagglutinin and the neuraminidase genes. Proc. Natl. Acad. Sci. USA 73:2142.[Abstract/Free Full Text]
  33. Fazekas de St. Groth, S., R. G. Webster. 1966. Disquisitions of original antigenic sin. I. Evidence in man. J. Exp. Med. 124:331.[Abstract]
  34. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY.
  35. Kavaler, J., A. J. Caton, L. M. Staudt, D. Schwartz, W. Gerhard. 1990. A set of closely related antibodies dominates the primary antibody response to the antigenic site CB of the A/PR/8/34 influenza virus hemagglutinin. J. Immunol. 145:2312.[Abstract]
  36. Clarke, S. H., L. M. Staudt, J. Kavaler, D. Schwartz, W. U. Gerhard, M. G. Weigert. 1990. V region gene usage and somatic mutation in the primary and secondary responses to influenza virus hemagglutinin. J. Immunol. 144:2795.[Abstract]
  37. Gerhard, W., A. M. Haberman, P. A. Scherle, A. H. Taylor, G. Palladino, A. J. Caton. 1991. Identification of eight determinants in the hemagglutinin molecule of influenza virus A/PR/8/34 (H1N1) which are recognized by class II-restricted T cells from BALB/c mice. J. Virol. 65:364.[Abstract/Free Full Text]
  38. Stark, S. E., A. J. Caton. 1991. Antibodies that are specific for a single amino acid interchange in a protein epitope use structurally distinct variable regions. J. Exp. Med. 174:613.[Abstract/Free Full Text]
  39. Scherle, P. A., W. Gerhard. 1986. Functional analysis of influenza-specific helper T cell clones in vivo. T cells specific for internal viral proteins provide cognate help for B cell responses to hemagglutinin. J. Exp. Med. 164:1114.[Abstract/Free Full Text]
  40. Kavaler, J., A. J. Caton, L. M. Staudt, W. Gerhard. 1991. A B cell population that dominates the primary response to influenza virus hemagglutinin does not participate in the memory response. Eur. J. Immunol. 21:2687.[Medline]
  41. Goodnow, C. C., J. Crosbie, H. Jorgensen, R. A. Brink, A. Basten. 1989. Induction of self-tolerance in mature peripheral B lymphocytes. Nature 342:385.[Medline]
  42. Smith, K. G., G. J. Nossal, D. M. Tarlinton. 1995. FAS is highly expressed in the germinal center but is not required for regulation of the B-cell response to antigen. Proc. Natl. Acad. Sci. USA 92:11628.[Abstract/Free Full Text]
  43. Lebecque, S., O. de Bouteiller, C. Arpin, J. Banchereau, Y. J. Liu. 1997. Germinal center founder cells display propensity for apoptosis before onset of somatic mutation. J. Exp. Med. 185:563.[Abstract/Free Full Text]
  44. Takahashi, Y., H. Ohta, T. Takemori. 2001. Fas is required for clonal selection in germinal centers and the subsequent establishment of the memory B cell repertoire. Immunity 14:181.[Medline]
  45. Adelstein, S., H. Pritchard-Briscoe, T. A. Anderson, J. Crosbie, G. Gammon, R. H. Loblay, A. Basten, C. C. Goodnow. 1991. Induction of self-tolerance in T cells but not B cells of transgenic mice expressing little self antigen. Science 251:1223.[Abstract/Free Full Text]
  46. Akkaraju, S., K. Canaan, C. C. Goodnow. 1997. Self-reactive B cells are not eliminated or inactivated by autoantigen expressed on thyroid epithelial cells. J. Exp. Med. 186:2005.[Abstract/Free Full Text]
  47. Lang, J., M. Jackson, L. Teyton, A. Brunmark, K. Kane, D. Nemazee. 1996. B cells are exquisitely sensitive to central tolerance and receptor editing induced by ultralow affinity, membrane-bound antigen. J. Exp. Med. 184:1685.[Abstract/Free Full Text]
  48. Hartley, S. B., C. C. Goodnow. 1994. Censoring of self-reactive B cells with a range of receptor affinities in transgenic mice expressing heavy chains for a lysozyme-specific antibody. Int. Immunol. 6:1417.[Abstract/Free Full Text]
  49. Snapper, C. M., H. Yamada, D. Smoot, R. Sneed, A. Lees, J. J. Mond. 1993. Comparative in vitro analysis of proliferation, Ig secretion, and Ig class switching by murine marginal zone and follicular B cells. J. Immunol. 150:2737.[Abstract]
  50. Martin, F., A. M. Oliver, J. F. Kearney. 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14:617.[Medline]
  51. Oliver, A. M., F. Martin, G. L. Gartland, R. H. Carter, J. F. Kearney. 1997. Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses. Eur. J. Immunol. 27:2366.[Medline]
  52. Oliver, A. M., F. Martin, J. F. Kearney. 1999. IgMhighCD21high lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells. J. Immunol. 162:7198.[Abstract/Free Full Text]
  53. Shih, T. A., M. Roederer, M. C. Nussenzweig. 2002. Role of antigen receptor affinity in T cell-independent antibody responses in vivo. Nat. Immunol. 3:399.[Medline]
  54. Hayakawa, K., M. Asano, S. A. Shinton, M. Gui, D. Allman, C. L. Stewart, J. Silver, R. R. Hardy. 1999. Positive selection of natural autoreactive B cells. Science 285:113.[Abstract/Free Full Text]
  55. Okamoto, M., M. Murakami, A. Shimizu, S. Ozaki, T. Tsubata, S. Kumagai, T. Honjo. 1992. A transgenic model of autoimmune hemolytic anemia. J. Exp. Med. 175:71.[Abstract/Free Full Text]
  56. Hannum, L. G., D. Ni, A. M. Haberman, M. G. Weigert, M. J. Shlomchik. 1996. A disease-related rheumatoid factor autoantibody is not tolerized in a normal mouse: implications for the origins of autoantibodies in autoimmune disease. J. Exp. Med. 184:1269.[Abstract/Free Full Text]
  57. Brodeur, P. H., R. Riblet. 1984. The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes. Eur. J. Immunol. 14:922.[Medline]
  58. Strohal, R., A. Helmberg, G. Kroemer, R. Kofler. 1989. Mouse V{kappa} gene classification by nucleic acid sequence similarity. Immunogenetics 30:475.[Medline]

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