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Original Article |
caton{at}wistar.upenn.edu
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Key Words: tolerance • autoantibodies • molecular mimicry • germinal center • CD4+ T cells
In addition to undergoing distinct differentiative fates in response to foreign antigens, B cells also appear to be heterogeneous in their responses to self-antigens. In recent years, studies using Ig transgenic (tg) mice have firmly established that although specificity of the BCR for a self-antigen can cause self-reactive B cells to be negatively selected from the primary B cell repertoire 1213, this is not absolute, as some B cells whose BCRs can react with a self-antigen can persist in the peripheral lymphoid organs of nonautoimmune mice 141516. Moreover, although elimination of autoreactive B cells from the primary repertoire has been analyzed in considerable detail, the processes that govern the fate of autoreactive B cells during memory formation remain obscure. As it is well established that germinal center formation depends on cognate interactions between antigen-specific B cells and CD4+ T cells, negative selection of autoreactive CD4+ T cells could limit autoreactive memory B cell formation 171819. However, self-reactive B cells activated by a cross-reactive antigen on a microbe would potentially have a source of linked CD4+ T cell help 2021. Furthermore, B cells responding to foreign antigens, through somatic hypermutation, could acquire reactivity with self-antigens 222324. Because of these possibilities, the germinal center has been proposed as a second window of BCR-mediated tolerance induction 1925. However, there is only indirect evidence that BCR specificity for self-antigens leads to negative selection of autoreactive B cells during memory formation 262728293031. Indeed, that autoreactive B cells can undergo clonal expansion and somatic mutation in certain B cell–mediated autoimmune disorders strongly suggests that autoreactive B cells can mature through the germinal center pathway 233233.
We have been examining how specificity for foreign and self-antigens affects B cell differentiation by using the influenza virus A/PR/8/34 (PR8) hemagglutinin (HA) as a well-characterized antigen 3435. We demonstrated previously that HA-specific B cells induced in response to PR8 virus immunization in BALB/c mice are composed of distinct populations that respond differently to antigenic stimulation by the HA. A major population of IgG-secreting ASCs dominates the primary response 5 d after immunization, and includes B cells that use a BCR clonotype (termed C12) that utilizes characteristic H chain variable (VH) and
Viruses and Immunizations.
Virus-specific ELISA.
Hemagglutination Inhibition Assay.
Enzyme-linked Immunospots.
Immunohistochemistry.
Hybridoma Generation.
Sequence Analysis of Antibody V Regions.
Flow Cytometry.
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After receptor engagement by antigen, B cells can undergo a variety of differentiative fates. In response to foreign antigens, mature B cells can become activated and differentiate into antibody-secreting cells (ASCs) and/or memory B cells. These differentiated populations have distinct functional potentials for postactivation events such as antibody production and somatic hypermutation, and appear to have developed along different pathways as indicated by their discrete anatomical locations and their differential expression of variable region clonotypes 123. In some cases, the pathway along which responding B cells develop can be influenced by signals mediated via the B cell receptor (BCR 4), yet there is also evidence that the preimmune repertoire contains separate populations of B cells that are predisposed to certain differentiative fates 567891011. The capacity for B cell populations to differentiate into either ASCs or memory B cells may play an important role in providing effective humoral immunity to complex infectious agents such as viruses. 
chain variable (Vk) gene segments 536. Several lines of evidence indicate that C12 B cells predominantly undergo terminal differentiation into IgG ASCs in response to virus immunization and that they do not participate in germinal center reactions in BALB/c mice 37. However, other clonotypes (including a clonotype termed C4) have been identified that are typically isolated among IgM-secreting hybridomas 5 d after immunization, and as somatically mutated and IgG-secreting hybridomas after secondary challenge 38. Further evidence of the distinct phenotypic potentials exhibited by these two populations of HA-specific B cells emerged with the observation that C12 B cells are negatively selected in tg mice that express the HA as a neo–self-antigen (HA104 mice), whereas HA-specific IgM-secreting B cells (including C4 B cells) were readily isolated after primary immunization 5. The finding that C4 B cells evaded negative selection from the primary B cell repertoire in HA104 mice despite their specificity for the neo–self-HA provided an opportunity to examine the extent to which mechanisms exist to mediate their negative selection during memory formation. We show here that HA104 and non-tg(BALB/c) mice generate equivalent HA-specific B cell responses after secondary immunization, and that HA-specific C4 B cells can undergo somatic hypermutation and clonal expansion in HA104 mice. In addition, we examine the role that CD4+ T cell help plays in the generation of autoantibody responses to the neo–self-HA.
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Mice.
HA104 and site 1 (S1)-specific TCR transgene (TS1) mice were described previously 5394041. Both lineages were backcrossed to BALB/c mice (Harlan) at least 7 and in most cases >10 generations before use in these experiments and were maintained in sterile microisolators at The Wistar Institute Animal Facility. In multiple experiments, non-tg littermates of HA104 mice generated equivalent ASC responses to BALB/c mice after virus immunization. Therefore, both non-tg and BALB/c mice were used in these experiments and are referred to as non-tg(BALB/c) mice.
Influenza viruses PR8 (A/Puerto Rico/8/34 [H1N1]), T3 (a mutant PR8 virus containing an aspartic acid to glycine interchange at amino acid 227 [35, 42]), and J1 (a reassortant of PR8 containing the nonserologically cross-reactive H3 HA [H3N1]; references 43, 44) were grown in the allantoic cavity of 10-d-old fertilized chicken eggs, purified by sucrose gradient centrifugation, and titered by chicken RBC agglutination 45. Mice were primed for memory B cell responses by immunization with 1,000 hemagglutinating units (HAU) intraperitoneally purified T3 or PR8 virus. For primary and secondary responses, mice were killed 5 or 3 d, respectively, after immunization with 1,000 HAU T3 or PR8 virus intravenously in 0.2 ml PBS. Secondary immunizations were performed a minimum of 4 wk after primary immunization.
ELISAs were done using purified T3, PR8, and J1 viruses as immunadsorbents as described previously 5. In brief, polyvinyl 96-well plates were coated with 25 µl diluted virus (1,000 HAU/ml in PBS plus azide). After incubation overnight at 4°C, plates were blocked with 1% BSA in PBS plus azide. Hybridoma culture supernatants or serum were added (diluted in 1% BSA in PBS plus azide) for 90 min. Bound antibody was detected by using alkaline phosphatase (AP)-conjugated goat anti–mouse IgG and/or IgM (diluted 1:1,000 in PBS plus azide; Southern Biotechnology Associates, Inc.) and developed using p-nitrophenyl phosphate. Absorbances were read at 405 nm using a microplate reader.
The ability of antibodies to neutralize viral agglutination of chicken RBCs was measured as described previously 45. In brief, 4 HAU of PR8 virus were plated (25 µl) in 96-well Multiscreen-HA plates (Millipore). Twofold dilutions of serum beginning with 1:100 were added (25 µl) to the virus and incubated for 1 h. 50 µl 1% RBCs was added and incubated for 30 min. Hemagglutination inhibition (HI) titers were determined as the highest serum dilution capable of inhibiting hemagglutination.
Virus-specific enzyme-linked immunospots (ELISPOTs) were done using purified T3, PR8, and J1 as described previously 5. In brief, 96-well Multiscreen-HA plates (Millipore) were coated with 50 µl diluted virus (1,000 HAU/ml in PBS). After overnight incubation (4°C), plates were blocked with 0.2 ml supplemented IMDM plus 5% FCS 40. Splenocytes were plated in triplicate in 0.2 ml at 1 x 106, 2.5 x 106, 6.25 x 105, and 1.25 x 105 cells/well for 4 h at 37°C. Bound antibody was detected with AP goat anti–mouse IgG and/or IgM (Southern Biotechnology Associates, Inc.). AP was developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in 0.1 M NaHCO3 plus 0.001 M MgCl2. ASC spots were counted using a dissecting scope.
Spleens were submerged in OCT (VWR Scientific), frozen with 2-methyl butane cooled with dry ice, sectioned, and fixed with acetone. Sections were stained as described 2. In brief, sections were blocked using PBS/2% BSA/0.1% Tween 20 and then stained with anti-IgD–biotin (Southern Biotechnology Associates, Inc.), peanut agglutinin (PNA)–horseradish peroxidase (HRP; EY Labs), and Extravidin-AP (Sigma-Aldrich) diluted in PBS/2% BSA/0.1% Tween 20. AP and HRP were developed using Fast-Blue BB base and 3-amino-9-ethyl carbazole (Sigma-Aldrich), respectively.
Hybridomas were generated from T3-immunized mice 3 d after secondary immunization by fusion with Sp2/0-Ag14 cells and subsequent selection with hypoaxanthine–azaserine as described previously 5. 10 d after fusion, hybridoma supernatants were aspirated and replaced with IMDM plus 10% FCS. Supernatants were screened 2 d later by ELISA for reactivity against T3 virus. T3-reactive hybridomas were expanded and their supernatants were screened for reactivity with T3, PR8, and J1 viruses and for H chain isotype.
Sequence analysis of hybridoma Ig H and L chain mRNA V regions was performed as described previously 5. In brief, RNA was isolated from 
106 hybridoma cells and reverse transcribed using a L chain and appropriate H chain C region–specific primer under standard conditions 46. PCR was carried out on the resulting cDNA using appropriate C chain and V region–specific primers: VH5'1 or VH5'2 to amplify H chain V regions and VkC4 to amplify L chains 547. Amplified products were run on a 1% agarose gel and purified using GeneCleanII (Bio101). Sequences were obtained using four-color dye chemistries with the ABI 373S sequencer (PerkinElmer) at the Nucleic Acid Facility at The Wistar Institute.
Flow cytometric analysis was performed on single cell suspensions made from pooled inguinal, brachial, axillary, and superficial cervical LNs on a FACScanTM flow cytometer (Becton Dickinson). 400,000–500,000 events were collected and analyzed using CELLQuestTM (Becton Dickinson). Antibodies used for staining were anti-CD4–FITC (GK1.5; BD PharMingen), anti-CD69–PE (BD PharMingen), and 6.5-biotin 41 detected by Streptavidin 670 (GIBCO BRL).
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HA104 and Non-tg(BALB/c) Mice Generate Equivalent HA-specific ASC Responses after Secondary Immunization.
HA104 mice express the influenza virus PR8 HA as a membrane-bound neo–self-antigen in a wide variety of tissues, including the bone marrow, spleen, and thymus 539. To examine how the neo–self-HA affects the ability of HA104 mice to generate HA-specific B cell responses, HA104 and non-tg(BALB/c) mice were immunized with the virus T3 (a virus identical to PR8 except for a single amino acid difference in a B cell epitope of HA [35, 42]). Using whole virus as an immunogen ensured that T cell help would not be limiting for HA-specific B cells, as T cells directed to other proteins in the virus particle could provide a source of intermolecular cognate help for HA-specific B cells 2048. Additionally, using T3 rather than PR8 virus to challenge the mice permitted an analysis of the fine specificity of the anti-HA antibody response (see below). 5 d after primary, or 3 d after secondary immunization, splenocytes from immunized animals were analyzed for the frequency of ASCs by ELISPOT (Fig. 1). We initially determined the frequency of ASCs that could react with the HA by comparing the frequencies that could react with PR8 virus with those that could react with the reassortant virus J1. J1 is identical to PR8 except for the presence of a serologically non–cross-reactive HA, and quantitates the frequency of ASCs that are directed to non-HA viral components (i.e., other virus proteins and components such as carbohydrates that derive from propagating the virus in hen eggs [43, 44]). The excess frequency of PR8-specific ASCs over J1-specific ASCs therefore indicates the number of HA-specific ASCs that were induced in response to virus immunization.
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To examine the magnitude of the HA-specific memory B cell response at the level of serum antibody, we measured the ability of serum antibodies obtained after secondary virus immunization of HA104 and non-tg(BALB/c) mice to inhibit hemagglutination (HI assay). The ability of antibodies to neutralize virus-induced hemagglutination in vitro requires B cell recognition of conformation-dependent epitopes on the HA and correlates with the ability of antibodies to protect against viral infection 4950. As shown in Fig. 1 E, the HI titers of serum from HA104 and non-tg(BALB/c) mice after secondary virus immunization were equivalent. Together, the findings indicate that secondary virus immunization induces memory B cell responses in HA104 and non-tg(BALB/c) mice that are of equivalent magnitude and that in each case are directed towards conformation-dependent epitopes on the HA molecule.
HA104 and Non-tg(BALB/c) Mice Do Not Differ in the Fine Specificity of Their Memory B Cell Response to T3 Virus.
As similar frequencies of HA-specific IgG ASCs could be activated from the memory B cell pool after a second exposure to virus in HA104 and non-tg(BALB/c) mice, this implied that HA-specific B cells are not negatively selected during memory B cell formation in HA104 mice. However, because B cell memory formation involves a series of poorly understood selection events that allow rare somatic mutants to preferentially expand and ultimately populate the memory pool 5152, we wanted to examine more closely whether HA-specific B cells are counterselected during the generation of the memory B cell pool. To this end, we examined the fine specificity of the response to T3 virus. The frequency of HA-specific B cells was related to those directed either to a non–self-epitope on the HA (the T3 mutation) or to other non-HA viral components (indicated by reactivity with J1). In this way, the frequency of B cells directed to T3 and J1, which provide a measure of those memory B cells specific for foreign (non-self) epitopes on the immunizing antigen, could be compared with the frequency with which anti–self-HA–specific B cells were generated in HA104 mice.
When the specificity of the primary response (Fig. 1 C) to T3 virus was examined in non-tg(BALB/c) mice, the majority of the ASCs were HA specific (i.e., did not react with J1 virus). A small excess of ASCs that could react with T3 but not with PR8 virus could also be detected, and corresponded to ASCs that recognize the mutant T3 epitope on the HA. In HA104 mice, the magnitude and the specificity of the primary IgM ASC response closely paralleled that of non-tg(BALB/c) mice. However, as described above, the frequency of IgG ASCs that could react with PR8 virus was decreased roughly fivefold relative to non-tg(BALB/c) mice. Moreover, because HA-specific IgG ASCs were negatively selected in HA104 mice, the relative proportion of the total IgG ASCs that were specific for the non–self-T3 epitope more than doubled in HA104 mice compared with non-tg(BALB/c) mice (64 vs. 27%). Therefore, at the level of the primary response, negative selection of HA-specific B cells altered the fine specificity of the IgG ASC response, which became skewed toward reactivity with the T3 mutation.
After secondary immunization (Fig. 1 D), the frequencies of IgM and IgG ASCs that could react with T3 virus increased substantially relative to the primary response, and were of equivalent magnitudes in HA104 and non-tg(BALB/c) mice. The IgM ASCs from HA104 and non-tg(BALB/c) mice reacted equally well with T3, PR8, and J1 viruses, indicating that these secondary response IgM ASCs were almost exclusively specific for non-HA epitopes on the virus particle. Non-HA epitopes include other viral epitopes as well as components that derive from propagating influenza virus in hen eggs, and it is possible that these secondary response IgM ASCs are directed to egg-derived carbohydrate molecules. Strikingly, the frequencies of T3-, PR8-, and J1-specific IgG ASCs were also similar in HA104 and non-tg(BALB/c) mice, and in each case roughly half of the IgG ASCs could react with T3 and PR8 but not with J1 and were therefore HA specific. Thus, unlike the primary response in which the IgG ASC responses in HA104 mice were predominantly directed toward the non–self-T3 epitope, memory B cell responses in HA104 mice were not skewed toward either the T3 mutation or non-HA epitopes on the virus particle.
To examine the specificity of the antibody response to T3 virus at the level of individual B cells, B cell hybridomas were generated 3 d after secondary immunization of HA104 and non-tg(BALB/c) mice and analyzed for their ability to react with T3, PR8, and J1 viruses (Table ). Consistent with the ELISPOT data, the overwhelming majority of the IgM-secreting hybridomas isolated from either HA104 or non-tg(BALB/c) mice were specific for non-HA viral components. The IgG-secreting hybridomas also closely paralleled the ELISPOT analysis, in that both the absolute numbers of HA-specific hybridomas isolated from HA104 and non-tg(BALB/c) mice, and the relative numbers of HA-, T3-, and J1-specific hybridomas were similar. Thus, based on the specificity of the B cell response to T3 virus, HA-specific B cells do not appear to be negatively selected during memory B cell formation in HA104 mice. Indeed, anti–self-HA–specific B cells did not appear to be at any disadvantage relative to either T3-specific, or to non-HA antiviral B cells in their ability to enter and be reactivated from the memory B cell pool.
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30% of the peripheral CD4+ T cells express high levels of the clonotypic TCR as detected with the anticlonotypic monoclonal antibody 6.5 41. In TS1 x HA104 mice, the majority of the clonotypic T cells are deleted during their development in the thymus 58. Nonetheless, a higher number of peripheral LN cells in TS1 x HA104 mice were detected by the 6.5 monoclonal antibody than were detected in non-tg(BALB/c) mice (1.1 ± 0.6 x 106 vs. 0.5 ± 0.4 x 106), indicating that some HA-specific CD4+ T cells evade deletion in TS1 x HA104 mice (Fig. 3 A). This conclusion is supported by recent studies in which we demonstrated that CD4+ T cells that evade deletion in TS1 x HA104 mice could be activated by S1 peptide in vitro and by PR8 virus in vivo 58. To examine whether the T cells that evade deletion in TS1 x HA104 mice might have interacted with the S1 peptide in vivo, freshly isolated LN cells from non-tg(BALB/c), TS1, and TS1 x HA104 mice were stained with anti-CD4, 6.5, and anti-CD69 antibodies and analyzed by flow cytometry (Fig. 3 B). In the LN cells from TS1 mice, the percentage of 6.5+ cells that were CD69+ was similar to the percentage of total 6.5lo cells that were CD69+ (7.6 vs. 7.8%). By contrast, the 6.5+ cells in TS1 x HA104 mice contained higher percentages of CD69+ cells than were found on 6.5lo cells (24.3 vs. 8.1%). As increased levels of CD69 are characteristic of antigen-experienced T cells 5960, the higher levels present on the autoreactive 6.5+ T cells suggest that these T cells had interacted with the S1 peptide in vivo.
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Although processes governing the negative selection of autoreactive B cells from the primary B cell repertoire have been extensively studied, how and if autoreactive B cells that evade negative selection from the primary B cell repertoire (or that acquire autoreactivity via somatic mutation) are regulated during memory formation has received little analysis. Because we found previously that separate populations of HA-specific B cells differed in their susceptibility to negative selection from the primary B cell repertoire in HA104 mice, we were able to evaluate whether a second window of tolerance induction might lead to the elimination of HA-specific B cells during memory B cell formation. We found that HA104 and non-tg(BALB/c) mice generated HA-specific memory B cell responses that were of equivalent magnitude. Moreover, the relative frequencies of memory B cells directed to the HA versus those directed either to a mutated epitope on the HA (the T3 mutation) or to non-HA epitopes on the virus (e.g., the nucleoprotein) were equivalent in HA104 and non-tg mice. Previous studies of antibody responses to species variants of self-proteins such as cytochrome c, hemoglobin, and MHC class II have demonstrated that negative selection of autoreactive B cells can focus the antibody responses toward epitopes that differ from the self-protein 626364. However, in these previous studies, the extent to which focusing of the response to non–self-epitopes reflected negative selection of autoreactive B cells from the primary repertoire versus during formation of the memory B cell pool was not established. We found that whereas negative selection of HA-specific B cells skewed the T3-induced primary response IgG ASCs toward specificity for the non–self-T3 mutation, this was not true for the memory response in HA104 mice. Thus, even though memory B cells are produced through stochastic processes that require the generation, selection, and expansion of mutated B cells 11515254, we did not find any evidence that HA-specific B cells in HA104 mice were at a disadvantage relative to B cells directed to epitopes other than HA on the same immunizing virus particle.
Why are HA-specific B cells not negatively selected during memory formation in HA104 mice? Antigen sequestration has been proposed as a reason that autoreactive B cells can avoid regulation by (or be "ignorant" of) self-antigens 65. However, three lines of evidence suggest that the HA is accessible to and can be recognized by HA-specific B cells in HA104 mice. First, the neo–self-HA is responsible for the negative selection of a major subset of HA-specific B cells from the primary B cell repertoire. Second, the neo–self-HA was able to activate HA-specific memory B cells from non-tg(BALB/c) mice when adoptively transferred with HA-specific T cells into HA104 mice, and this activation led to ASC formation and germinal center development in the spleens of recipient mice. Third, the neo–self-HA was able to stimulate autoantibody production in HA104 mice that had been mated with mice transgenic for an HA-specific CD4+ TCR. Moreover, the autoantibodies elicited by the neo–self-HA were able to recognize conformation-dependent epitopes on the viral HA molecule. Based on these lines of evidence, neither antigen inaccessibility nor incorrect folding of the neo–self-HA appears to be likely explanation for the ability of autoreactive HA-specific memory response B cells to develop in HA104 mice. There is also evidence that the presence of autoantibodies may mask self-antigens and thus limit the accessibility of the antigens to self-reactive B cells 18. In the case of HA-specific antibodies elicited by acute viral immunization, masking of HA by these antibodies may indeed allow for autoreactive B cells that emerge after immunization to escape tolerance induction mechanisms. However, that we were able to measure autoantibody production in naive TS1 x HA104 mice suggests that masking of autoantigens by virus-induced antibodies is not necessary for the initiation of autoantibody production to HA.
An alternative explanation for the HA-specific memory B cell responses that could be induced in HA104 mice is that B cells are not subjected to negative selection during memory formation based on the specificity of their BCR for a self-antigen. Instead, the availability of CD4+ T cell help may play a crucial role in regulating memory autoantibody responses. Germinal center reactions depend on cognate interactions between antigen-specific B cells and CD4+ T cells 17, and it has long been recognized that negative selection of autoreactive CD4+ T cells could play an important role in regulating autoantibody responses 186667. Yet, because of the possibility that a foreign antigen (such as a virus) could contain B cell epitopes that cross-react with self-antigens and provide autoreactive B cells with help directed to non–self-proteins 2021, several studies have examined whether B cell intrinsic tolerance mechanisms exist to prevent the development of autoreactive B cells in germinal centers. Evidence in support of such mechanisms is mostly indirect, and has been derived from examining the effects of administering high concentrations of soluble antigens to germinal center B cells and/or examining how genes that affect lymphocyte survival influence germinal center reactions 252728293031. We demonstrated here that clonally expanded and somatically mutated B cells using a clonotype (C4) that is typical of PR8 HA–specific memory B cell responses of influenza virus–immunized mice BALB/c mice could be isolated from virus-immunized HA104 mice. Because clonal expansion and somatic hypermutation occur predominantly in germinal centers 5154, this provides direct evidence that autoreactive HA-specific B cells had developed in the germinal center pathway. In addition, two observations suggest that HA-specific antibodies also underwent affinity maturation in HA104 mice. First, HA104 and non-tg(BALB/c) mice contained similar frequencies of HA-specific ASCs. Second, sera from HA104 and non-tg(BALB/c) mice contained similar titers of hemagglutination-inhibiting antibodies. Unless ASCs from HA104 mice secreted more antibody per ASC than did those in non-tg(BALB/c) mice (for which there is no precedence), equivalent serum titers of HI antibodies suggest that the overall affinity for the HA of serum from HA104 mice is comparable to that of non-tg BALB/c mice. Therefore, to the extent that the levels of hemagglutination-inhibiting antibodies that are present in the sera of non-tg(BALB/c) mice after secondary immunization reflect the outcome of somatic mutation and affinity maturation 38, that HA104 and non-tg(BALB/c) mice contain both similar frequencies of HA-specific ASCs and comparable levels of hemagglutination-inhibiting serum antibodies suggests that HA-specific antibodies also undergo affinity maturation in HA104 mice. In this regard, it is noteworthy that a low affinity for self-antigens has been shown previously to allow B cells to evade negative selection from the primary repertoire 68. As HA104 and non-tg(BALB/c) mice differ substantially in the frequency of HA-specific IgG ASCs that are activated after primary immunization, it is difficult to assess the relative affinity of their primary serum responses. However, if the HA-specific B cells that evade negative selection from the primary B cell repertoire in HA104 mice are of relatively low affinity for the HA, the studies here suggest that affinity maturation allows the secondary response to achieve an affinity comparable to those in non-tg(BALB/c) mice.
Finally, it is significant to note that the ability of HA-specific C4 B cells to develop in the germinal center pathway in HA104 mice was dependent on virus immunization. Whereas HA-specific C4 B cell hybridomas isolated 3 d after secondary immunization in HA104 mice were somatically mutated and had undergone H chain class switching, those isolated 5 d after primary immunization of HA104 mice expressed unmutated IgM 5. This requirement for virus immunization most likely reflects a need for cognate T cell help to permit activation of these autoreactive B cells. We have demonstrated previously that HA-specific CD4+ T cells are negatively selected in HA104 mice 69, and the idea that negative selection of HA-specific CD4+ T cells plays a role in regulating autoantibody production in HA104 mice was also inferred from studies here using TS1 x HA104 mice. Even though HA-specific T cells undergo negative selection during their development in TS1 x HA104 mice 58, the rare autoreactive CD4+ T cells that evade deletion by the neo–self-HA are sufficient to induce autoantibody production in TS1 x HA104 mice. It will be interesting in future experiments to determine whether the HA-specific autoantibodies in TS1 x HA104 mice derive from B cells that would have otherwise undergone deletion from the primary repertoire or from HA-specific B cells that were induced to form memory by the neo–self-HA in the presence of persistent T cell help. Regardless of the source of autoantibodies in TS1 x HA104 mice, it is clear that HA as a neo–self-antigen is capable of being recognized by HA-specific B cells in vivo. Moreover, the findings presented here provide evidence that provision of CD4+ T cell help can play a crucial role in the activation of autoreactive B cells.
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This work was supported by National Institutes of Health grants 5T32EY07131, AI24541, and CA10815.
Submitted: 23 June 2000
Revised: 18 September 2000
Accepted: 13 October 2000
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