Generation and selection of an IgG-driven autoimmune repertoire during B-lymphopoiesis in Igµ-deficient/lpr mice

Jane Seagal, Efrat Edry, Hadas Naftali and Doron Melamed1

Department of Immunology, Bruce Rappaport Faculty of Medicine and 1 Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa, Israel

Correspondence to: D. Melamed; E-mail: melamedd{at}techunix.technion.ac.il
Transmitting editor: D. Wallach


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Class switch recombination (CSR) is a well-regulated process that occurs in peripheral lymphoid tissue, and is thought of as an important factor constructing the memory repertoire. We have recently shown that CSR normally occurs during bone marrow (BM) development, and these isotype-switched B cells are negatively selected by Fas signaling. This novel pathway of B cell development may generate a primary repertoire driven by {gamma}-heavy receptors, the nature of which is yet unknown. To study this {gamma}H-driven repertoire we used mice lacking IgM-transmembrane tail exon (µMT), where B cell development is limited by their ability to undergo CSR. We already showed that lack of Fas signaling rescues development of a significant population of isotype-switched B cells and production of high titers of non-IgM serum antibodies in µMT mice deficient in Fas (µMT/lpr), thereby providing a mouse model allowing the assessment of {gamma}H-driven repertoire. Using a tissue array and phage display epitope library we report here that IgG repertoire in µMT/lpr mice is oligo-monoclonal, bearing self-tissue reactivity. This is supported by analysis of the V{kappa} utilization in peripheral B cells from µMT/lpr mice, which revealed a strikingly restricted repertoire. In contrast, µMT/lpr B cells that are grown in non-selective BM cultures utilize a wide repertoire. These results suggest that the Fas pathway is an important regulator in the generation and selection of an autoimmune {gamma}H-driven repertoire in vivo.

Keywords: antibodies, autoimmunity, B lymphocytes, hematopoiesis, repertoire development


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
B cell development in the BM is guided by successive V(D)J recombination and is driven by the expression of µH chain. Together with the surrogate light chain, µH constructs the preBCR, which signals for proliferative expansion of proB cells. At the immature stage, µH pairs with the light chain to form the IgM receptor, which signals for negative and positive selection of B cells (13). Hence, B cell hematopoiesis depends on µH expression and signaling, as the development of B cells in mice bearing signaling mutation or incapable of expressing µH (µMT mice) is blocked or severely impaired [reviewed in (4,5)].

Earlier studies have shown that IgD can substitute for IgM in promoting B cell development (6). In contrast, studies with IgG transgenic mice show that {gamma}H receptors are not efficient in promoting B cell development [reviewed in (7)]. This is thought to result from differences in BCR proximal signaling residues relative to the µH (7,8). However, {gamma}H receptors are superior in signaling for B cell proliferation and burst enhancing of plasma cell formation, suggesting that {gamma}H expression is a molecular determinant of a memory B cell and a memory response (9). Interestingly, some IgM-expressing memory B cells have been described in human and mouse (10,11).

Despite its inefficiency in promoting B cell development in transgenic mice, several studies suggested that isotype switching to {gamma}H receptor can occur at early stages of B cell development (12), and spontaneously in transformed B cell lines (13). Recently we showed that class switch recombination (CSR) normally occurs during B cell development and that isotype-switched B cells are sensitive to Fas-mediated apoptosis (14). Using the µMT mouse model, where B cell development is blocked at the proB stage due to targeting of a stop codon in the transmembrane µH exon (15), we and others have shown that extended survival or intestinal immunization can rescue development of some isotype-switched B cells (1618). This implies that {gamma}H receptors can support B cell development, but the extent of this developmental pathway is unknown. Furthermore, the primary repertoire encoded by these {gamma}H-driven receptors and their tolerance requirements are unknown, relative to the well-described µH receptors. This is particularly important since expression of {gamma}H receptors may be sufficient to allow B cell development into the memory compartment (9,14), effectively circumventing peripheral tolerance requirements. This notion is supported by studies showing that tolerance is maintained in anti-self IgM Tg mice (19,20), whereas autoantibodies are produced in anti-self IgG transgenic mice (21,22).

In a previous study we showed that unlike µH-expressing B cells, {gamma}H-expressing cells are controlled by Fas signaling, as µMT mice deficient of functional Fas (µMT/lpr) allow significant development of isotype switched B cells in vivo and in vitro (14,17). These mice developed anti-chromatin antibodies and failed to respond to T-dependent and T-independent antigens (17). Since development of µMT B cells is limited by isotype switching, this mouse can be used as a perfect model to study the {gamma}H repertoire formation and its tolerance requirement. Here, we bring data showing that {gamma}H-driven repertoire, developing in µMT/lpr mice, is oligo-monoclonal and bears significant tissue reactivity. Furthermore, using BM culture system we bring evidence that this oligo-monoclonal self-reactive repertoire develops through a high process of selection. Hence, we propose that Fas regulates the {gamma}H-driven repertoire and the generation of B cell autoimmunity.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Normal C57/BL6 (B6), C57/BL6-Faslpr/Faslpr (B6/lpr) and C57/BL6-µMTMT (µMT) (15) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice carrying both lpr and µMT homozygous mutations (µMT/lpr) were generated by crossing B6/lpr and µMT mice.

BM cell culture and FACS analysis
IL-7-driven BM cultures for growth of B cell precursors from normal and µMT/lpr mice were prepared as we have previously described (23). In some experiments BM cells were treated to remove Ig+ B cells on day 0 using magnetic beads (Miltenyi Biotec, Auburn, CA). Cells grown in cultures were stained for the indicated surface markers using fluorescently labeled antibodies and analyzed by FACS as we have described (23). In some experiments, spleen cells were stained for the indicated surface markers and analyzed by FACS.

Cell sorting
Ig{kappa}/{lambda}+ and Ig{kappa}/{lambda}neg splenic and BM culture B cells were sorted using MACS microbeads (Miltenyi) and polyclonal goat anti-mouse {kappa} and goat anti-mouse {lambda} antibodies (Southern Biotechnology Associates, Birmingham, AL). Sorted cells were visualized using streptavidin-FITC and analyzed by FACS, or were used for mRNA preparation.

Analysis of serum Ig and plasma cells
ELISPOT assays to detect plasma cells were performed as described previously (24). For the spot assay we used spleen cells from normal or from µMT/lpr mice, before or after depletion of Ig+ cells.

Nucleic acid analysis
Total RNA prepared from sorted Ig{kappa}/{lambda}+ and Ig{kappa}/{lambda}neg cells was reverse transcribed to cDNA and subjected to PCR using degenerate V{kappa} framework-3 (FW3) and C{kappa} primers as described (17). The resulting V{kappa}-C{kappa} products were cloned and single colonies were sequenced to determine the V{kappa} and the J{kappa} used in each, using GenBank and IgBlast database. Productiveness of V{kappa}-J{kappa} rearrangements was determined based on the presence of conserved amino acid residues as described (25). In some experiments RT–PCR amplification of mRNA samples for IgG1 germline and post switch transcripts as well as for activation-induced cytidine deaminase (AID) was carried out using the same conditions and primer sequencing as described (26).

Tissue array
Normal mouse tissue samples were homogenized in lysis buffer containing 1% NP-40, 0.1% sodium desoxycholate, 0.01% SDS in PBS and protease inhibitors, followed by incubation for 30 min at 4°C. After centrifugation, cleared tissue lysates were analyzed by SDS–PAGE on 12% gel, transferred to nitrocellulose membrane blots and blocked with 5% milk. Membranes were then incubated with sera collected from µMT/lpr or normal B6/C57Bl mice (diluted 1:500 to 1:1500). HRP conjugated goat anti-mouse IgG (Jackson) was used for detection. Visualization of specific bands was performed by ECL reaction.

Screening of phage display epitope library with µMT/lpr serum
Sera from µMT/lpr mice were used to screen a 12-mer phage-display epitope library, which has previously been described (27). Positive phages were isolated, collected and sequenced as described (28). To determine binding affinity, positive phages were adjusted to the same concentrations and equal amounts were applied to nitrocellulose membranes. Membranes were then exposed to serial dilutions of each serum, followed by incubation with anti-mouse IgG-HRP. The optical density was determined by scanning of the films and densitometric analysis of ECL signals. For each phage, optical density was plotted versus the respective IgG concentration in serum (in molar). Affinity was determined as the concentration in molars obtained for optical density values that are 50% relative to those representing maximal binding.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Class switch recombination occurs during µMT/lpr B cell development in vitro
We have previously shown that CSR spontaneously occur in normal and µMT B cell development, in vivo and in vitro, but these cells are negatively selected by Fas signaling (14). In µMT/lpr mice, unlike in µMT mice, lack of functional Fas allows development and maturation of isotype-switched B cells. To confirm that CSR normally occurs in developing µMT/lpr mice we utilized our BM culture system, which allows preferential growth of B cells in a non-selective in vitro environment. To account for isotype switching in the culture we performed RT–PCR for detection of IgG1 germline and post-switch transcripts and AID expression as we have described (14); signals indicative of ongoing CSR (26). To exclude the possible contribution of Ig+ memory B cells that may be found in BM, cultured cells were depleted of Ig+ cells on day 0 (14). The results in Fig. 1(A) clearly show that CSR normally occurs in normal and in µMT/lpr BM cultures, as significant expression of IgG1 germline and post-switch transcripts and AID were evident. In control cultures of normal splenic B cells, class switching was undetected, but could be induced upon LPS treatment as has been described (29). Further staining for surface IgG expression revealed a significant population of IgG-expressing cells in both cultures (Fig. 1B). We conclude that as found in normal and µMT B lymphopoiesis, CSR spontaneously occur in µMT/lpr BM cultures. It is yet to be determined whether CSR frequency is not increased in our BM culture conditions relative to that occurring in the BM in vivo.



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Fig. 1. Ongoing isotype switching in normal and µMT/lpr BM cultures. (A) mRNA samples from BM cultures of normal and µMT/lpr mice were subjected to RT–PCR amplification for IgG1 germline and post-switch transcript as well as for AID expression. Normal splenic cells either unstimulated or stimulated with LPS were used as negative and positive controls, respectively. (B) BM cells grown in culture were stained for CD19, B220 and IgG and analyzed by FACS. All the results shown are representative of 3–5 mice in each group.

 
Serum antibodies from µMT/lpr mice are reactive to self-tissue antigens
The µMT/lpr mice develop high titers of serum Ab rapidly after birth but fail to respond to exogenous T-dependent or T-independent antigen (17). Sera collected from these mice exhibited anti-chromatin reactivity, but at the same time were extremely heterogeneous in heavy and light chain isotypic distribution (17,24). To better probe for autoimmune reactivity of the {gamma}H-driven repertoire developed in µMT/lpr mice we used a mouse tissue array. All normal serum samples tested (in dilutions of 1:500 up to 1:1500) provided the same pattern of bands, probably corresponding to IgH and IgL found in these tissues as well as general cross-reactivity (Fig. 2, top panel). However, in tissue arrays where serum samples from µMT/lpr mice were applied, additional bands were detected in one or more of the assayed tissues. Remarkably, each of these µMT/lpr serum samples had specific reactivity with a different tissue (Fig. 2, middle panel, reactivity to muscle and heart tissues; Fig. 3, bottom panel, reactivity to thymus, lung, muscle, spleen and skin). The fact that a specific serum is recognizing the same band in several tissues may suggest that this antigen is present in all of these tissues, or that it reflects cross reactivity between similar tissue antigens. Such tissue reactivity was identified in ~50% of the analyzed µMT/lpr sera and was obtained for adult mice (>3 months). It therefore suggests that µMT/lpr mice develop a limited autoimmune repertoire bearing differential tissue specificity, which is uniquely obtained for individual mice. This autoimmune repertoire is driven by {gamma}H antigen receptor.



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Fig. 2. Self-reactivity of µMT/lpr sera in tissue array. Serum samples from normal and µMT/lpr mice were tested in a tissue array for tissue reactivity as described in Methods. Shown are representative blot arrays obtained for a normal (top panel) and two different µMT/lpr serum samples (middle and bottom panels). Tissue specific bands identified by µMT/lpr sera are shown by an arrow. The results represents five normal serum samples and 10 different µMT/lpr samples. Tissues order is: 1, thymus; 2, liver; 3, lung; 4, muscle; 5, spleen; 6, kidney; 7, skin; 8, heart; 9, brain.

 


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Fig. 3. Sorting Ig+ cells separates splenic B cells from plasma cells. Kappa and lambda-expressing cells from spleens of normal and µMT/lpr mice were sorted using magnetic beads. (A) Histograms showing the relative frequency of {kappa}/{lambda} cells before sorting, in the positive and in the negative populations. (B) Sorted cells were cultured on filters and plasma cells were visualized by ELISPOT assay. Shown are representative results from three different mice in each group. (C) Syndecan-1 expression in spleen cells from normal (top) and µMT/lpr mice gated for CD19+Ig+ (left panel) or CD19+/Igneg (right panel). Histograms shown are representative of at least four mice from each group.

 
Serum antibodies from µMT/lpr mice recognize an immunodominant epitope
To further study the limited µMT/lpr antibody repertoire we used a phage display 12-mer amino acid epitope library. This library is set to detect mimotopes only if the relative concentration of specific antibodies is above a certain threshold. Thus, a normal (specific pathogen free) mouse serum fails to detect any phage, whereas sera from an immunized mouse and from HIV patients efficiently detect multiple mimotopes [J. Gershoni, personal communication and (28)]. Serum samples from individual µMT/lpr mice were used for this assay and 20–30 different positive phages were picked for each serum. Sequencing analysis of the mimotopes revealed that 80–95% of the phages recognized by each serum included specific motifs consisting of 5–6 amino acids (Table 1). Phages with reduced affinities had amino acid substitutions at the motif site as indicated (Table 1). Remarkably, every µMT/lpr serum tested revealed a different motif sequence, and cross reactivity between different µMT/lpr sera was not found (data not shown). Additionally, none of the picked phages reacted against normal mouse serum (data not shown). The possibility of picking the same phage is unlikely, as all picked phages had different nucleotide sequences before and after the motif, and due to the complexity of this library (>109). These results suggested that µMT/lpr mice develop a unique oligo-monoclonal autoimmune repertoire.

An oligo-monoclonal B cell population is selected in µMT/lpr mice
The results obtained by the epitope display library prompted us to confirm whether µMT/lpr mice develop an oligo-monoclonal B cell population. To do so we performed analysis of the {kappa}L chain repertoire that is utilized by µMT/lpr splenic B cells, as well as the J{kappa} usage. For this analysis, mRNA samples from individual mice were subjected to RT–PCR amplification using consensus V{kappa} FW3 and C{kappa} oligos. PCR products were cloned and sequenced and 30–50 sequences bearing productive V{kappa}-J{kappa} rearrangement were considered for the analysis for each sample. To eliminate over representation of plasma cell mRNA, Ig+ B cells were sorted by magnetic beads using anti {kappa}/{lambda} reagents, whereas plasma cells, which are not expressing surface Ig, remained in the {kappa}/{lambda}neg population. Sorted cells were highly purified in the normal mouse (99%) but less purified for the µMT/lpr (~80–85%) (Fig. 3A). However, considering that Ig+ B cells in the µMT/lpr are <5%, this purification degree reflects a 16-fold increase in their purity. Nevertheless, we confirmed absence of plasma cells in both of the sorted cell populations (normal and µMT/lpr) by ELISPOT assay. As shown in Fig. 3B, sorted {kappa}/{lambda}+ cells from both normal and µMT/lpr spleens had essentially no plasma cells, whereas most of the plasma cells remained in the {kappa}/{lambda}neg population. Lack of soluble Ab in the {kappa}/{lambda}+ cultures was also confirmed by ELISA and western blots (data not shown). In addition, the plasma cell marker syndecan-1 is expressed in the CD19+/Igneg but not in the sorted CD19+/Ig+ splenic cell populations in both the normal and the µMT/lpr mice (Fig. 3C).

The results of the sequencing analysis for the sorted {kappa}/{lambda}+ B cells and the {kappa}/{lambda}neg populations for individual mice are shown in Fig. 4. In Ig+ B cells from a normal mouse a wide repertoire can be found contributed to by many V{kappa} genes (Fig. 4A, left panel), and all J{kappa} segments are used (Fig. 4B). In striking contrast, we found an oligo-monoclonal B cell population that is preferentially selected in µMT/lpr mice. Sequencing analysis revealed that 90–100% of the sequences determined for the µMT/lpr Ig+ B cells had utilized the same V{kappa}, and predominantly used the downstream J{kappa}4 or J{kappa}5 segment (Fig. 4C). Remarkably, and in support of our epitope display assay, in each µMT/lpr mouse a different V{kappa} was utilized in the selected Ig+ oligo-monoclonal B cell population in the spleen. Analysis of the plasma cell repertoire (the {kappa}/{lambda}neg population) for the same mice revealed dominance of the same V{kappa} gene identified in the corresponding Ig+ cells, thereby reflecting expansion of the same B cell clones (Fig. 4C).



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Fig. 4. Ig{kappa} repertoire and J{kappa} utilization in sorted normal and µMT/lpr splenic B cells. V{kappa} and the J{kappa} usage in sorted cells from spleen of the indicated mice was determined as described in Methods. For each sample, 30–50 sequences bearing in-frame rearrangement were considered for the analysis. (A) Relative frequency of V{kappa} found in sorted Ig+ cells from a normal mouse. (B) Usage of J{kappa}1 and J{kappa}2 relative to J{kappa}4 and J{kappa}5 in sorted Ig+ cells from a normal mouse. (C) Individual sequencing analysis of V{kappa}-J{kappa} joints from Ig+ and Igneg spleen cells from two representative µMT/lpr mice. For each sequence, shown are the V{kappa} gene and the J{kappa} segment that are used and the relative frequency. Amino acid residues shown in bold are conserved and important in determining rearrangement productiveness. Results are represent four different experiments.

 
Wide V{kappa} repertoire is generated in developing µMT/lpr B cells
To test whether the oligo-monoclonal B cell repertoire obtained in µMT/lpr mice reflects a high process of selection rather than a preferential recombination of particular V{kappa} genes, we utilized our non-selective BM culture system. To analyze the V{kappa} repertoire in the non-selected µMT/lpr Ig+ B cells grown in culture we used anti-{kappa} and anti-{lambda} antibodies to sort all Ig+ cells using magnetic beads. In µMT/lpr cultures we usually detect 3–10% of Ig+ B cells [Fig. 5A, left panel and (14)], but sorting for {kappa}/{lambda}+ cells increased the frequency by at least 10–15-fold resulting in ~80% of {kappa}+ cells (Fig. 5A, right panel). RT–PCR analysis for V{kappa}-C{kappa} revealed that such mRNA is essentially not detected in the Igneg B cell precursors (Fig. 5B), thereby confirming early in vivo observations showing very low frequencies of light chain recombination in developing B cells that fail to rearrange and express heavy chain (15). Sequences of V{kappa}-J{kappa} joining of sorted cells were determined following RT–PCR amplification as detailed before. For each mouse we analyzed 30–50 sequences containing productive V{kappa}-J{kappa} rearrangements. The results obtained clearly show that Ig+ µMT/lpr B cells that develop in a non-selective BM culture environment utilize a wide V{kappa} repertoire. In contrast to the oligo-monoclonal mature repertoire in vivo, we found that many V{kappa} genes and all J{kappa} segments contribute to generate a wide random repertoire in µMT/lpr Ig+ B cells that are grown in BM cultures (Fig. 5C). Interestingly, the J{kappa} utilization in these cells was significantly biased towards downstream J{kappa}4 and J{kappa}5 (80–90%). This observation suggests ongoing light chain rearrangements in the µMT/lpr BM culture. Since BCR expression and light chain allelic exclusion are limited by the occurrence of isotype switching, these cells probably continue to rearrange the kappa locus, resulting in increased utilization of downstream J{kappa}. Thus, we conclude that a wide repertoire is constructed during the development of µMT/lpr B cells and that a strict selection process allows maturation and survival of a particular oligo-monoclonal population. The fact that such cells are not developing in Fas-sufficient µMT mice suggests that this selection is controlled by the Fas pathway.



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Fig. 5. A wide kappa light chain repertoire is generated in developing µMT/lpr B cells in BM cultures. µMT/lpr BM cultures were prepared as described. On day 5, Ig+ cells were sorted using anti {kappa}/{lambda} antibodies and magnetic beads. (A) A representative FACS analysis of µMT/lpr BM culture before and after sorting. Shown is surface kappa expression in B cell precursors of unsorted (left panel) and sorted (right panel) cell populations. (B) RT–PCR analysis for V{kappa}-C{kappa} in sorted {kappa}/{lambda}neg and {kappa}/{lambda}+ cell populations. mRNA from sorted cells was RT–PCR amplified for V{kappa}FW3-C{kappa} or for actin as described. Shown are results of two sorting experiments. (C) mRNA from sorted cells was RT–PCR amplified using degenerate V{kappa} FW3 and C{kappa} primers. PCR products were cloned and sequenced to determine the V{kappa} and J{kappa} usage in the sorted cells as described in Methods. Shown are results from the same two mice analyzed in Fig. 5 for the peripheral B cell repertoire. For each mouse (top and bottom panels) the V{kappa} repertoire is shown in a pie plot and the J{kappa} usage in a histogram. The results are representative of total of four different mice.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
BCR specificity is a major factor determining selection of primary repertoire, and is thought to protect from the onset of B cell autoimmunity (1,3,3032). Positive and negative signals participate to construct and shape this repertoire and survival of the B cells (2,33). It is generally accepted that this wide repertoire is driven by µH BCR (1,2). However, little is known, if anything, about developmental pathway and repertoire formation that is driven by {gamma}H BCR. In µMT mice, B cell maturation is limited to cells that have successfully undergone isotype switching (1618). Hence, this mouse model is particularly useful in studying roles for the development selection and repertoire of {gamma}H-expressing B cells.

An important question that needs to be addressed is whether {gamma}H-expressing B cells can actually develop in the BM. This is particularly important since many studies have shown severe impairment of B cell development and increased autoimmunity in {gamma}H transgenic mice (7,8). An alternative possibility, as we have recently shown both in vivo and in vitro, is that isotype-switched developing B cells are negatively selected by Fas signaling (14). It has been well established that µMT B cell precursors can undergo isotype switching and upon extended survival, such as in the absence of functional Fas (17) or overexpression of bcl2 (16), undergo maturation and secrete non-IgM serum Ig. Isotype switching to IgA in µMT mice can also be induced upon intestinal stimulation (18) and may also be affected by genetic background (34). The data we present here clearly show that class switching spontaneously occurs in µMT/lpr BM cultures, thereby implicating that CSR is a biologically relevant process in µMT/lpr B hematopoiesis. The fact that such cells are not developing in µMT mice in vivo (15) suggests that isotype-switched B cells are negatively selected by Fas signaling. Thus, in addition to the developmental disadvantage of {gamma}H-expressing B cell precursors, such cells may confer a potential risk and are therefore deleted by Fas/FasL pathway. Although we found a relatively high frequency of CSR in vitro, we cannot exclude the possibility that CSR in vitro occurs at a higher frequency than in the BM in vivo. Studies elucidating this question are now being conducted.

IgM and IgG receptors utilize distinct signaling pathways (35), and IgG is thought to be a molecular determinant of memory response (9). In mature IgM-expressing B cell pool, CSR is limited by availability of T-cell help, a peripheral mechanism to maintain self-tolerance, and is thought to occur during the germinal center response (1). Hence, CSR in B lymphopoiesis may circumvent this peripheral tolerance mechanism and confer severe autoimmune risk (14). This is supported by recent studies showing that generation of IgG+ memory B cells (36) and acquisition of somatic mutations (37) can occur independently of germinal center formation, and that isotype switching can rescue anergic self-reactive B cells to secrete autoantibodies (38). Hence, elimination of {gamma}H-expressing cells in the BM by Fas signaling provides this death pathway with a major function in protection from autoimmunity, particularly since IgG+ memory B cells are not sensitive to Fas-mediated apoptosis (39). This is supported by showing that µMT/lpr mice have increased frequency of plasma cells (24) and that µMT/lpr Ig+ B cells express the marginal zone phenotype CD21+/CD23– (14).

Despite the fact that VDJ recombination in µMT mice is intact (15), lack of functional Fas results in the generation of an autoimmune oligo-monoclonal B cell repertoire in the periphery (Fig. 4). This can explain the lack of response to external antigens by µMT/lpr mice (17). In contrast, µMT/lpr B cells developing in vitro in a non-selective environment utilize a wide repertoire of receptors, suggesting that a high process of selection of autoreactive clones takes place in the µMT/lpr mice. In B6/lpr and MRL/lpr mouse models an autoimmune repertoire (mainly IgG) develops with age (40). Earlier studies have shown that these mice develop a significantly skewed repertoire with the progression of the diseases, but have a normal repertoire at young ages (41), thereby allowing responsiveness to external T-dependent and T-independent antigens (17). A similar skewed antibody repertoire has been demonstrated in SLE patients (42,43). However, in both lpr mouse models and in SLE patients this skewed repertoire develops from a wide pre-existing repertoire during the progression of the disease. In µMT/lpr mice both Ig+ B cell repertoire and plasma cell repertoire are identically oligo-monoclonal, implicating the preferential survival and expansion of particular B cell clones. In contrast to the wild-type lpr mice and SLE patients, this oligomonoclonaly develops from a limited repertoire, as µMT/lpr mice also fail to respond to external antigen at any tested age [(17) and our unpublished data]. In addition, the µMT/lpr autoimmune repertoire is developing in a {gamma}H-driven pathway, whereas in the wild-type lpr mice and SLE patients it is thought that such a repertoire originates from a wide IgM repertoire that undergoes polyclonal activation and selection (44,45). Thus, lack of Fas in the µMT/lpr mice may not only allow survival of isotype-switched B cells but also promotes the selection and generation of an oligo-monoclonal self-reactive repertoire. Such a role of Fas in selection of self-reactive IgM-expressing B cells in the periphery was also demonstrated in earlier studies showing that Fas signaling has an important role in promoting selection and activation of self-reactive IgM-expressing cells and in memory formation (37,46,47). Thus, Fas signaling is involved in selection of autoreactive IgM- and IgG-expressing B cells. The fact that each µMT/lpr mouse develops a unique autoimmune monoclonal repertoire suggests that self-reactivity is required for selection but that the target self-antigen may have some degree of randomness. As mice bearing the µMT mutation may have abnormal environmental effects in modulating B cell development and selection, it is now important to directly show a role of Fas in selection of IgG+ developing B cells in a normal mouse.

The remarkable observation of an individual monoclonal repertoire developed in µMT/lpr mice was evident by both phage display epitope library and sequencing of V{kappa} repertoire. This may suggest that autoimmunity in this mouse model is not directed to particular tissue, as also revealed by our tissue array. Interestingly, all V{kappa} genes identified in the µMT/lpr mice utilized predominantly J{kappa}4 or J{kappa}5. Similarly, increased utilization of downstream J{kappa} was also obtained in our BM cultures of µMT/lpr mice (Fig. 5). The utilization of the downstream J{kappa}s may reflect ongoing Ig{kappa} rearrangements, reminiscent of receptor editing (30,48). Since a limiting factor in the development of these cells is the occurrence of isotype switching, it is possible that light chain recombination initiates and continues due to the failure to express a BCR and to establish allelic exclusion. This is supported by earlier studies showing that B cells undergoing receptor editing stay for extended duration in the BM (49), and that light chain rearrangements may precede or initiate at the same time as heavy chain rearrangements (50). Also, V{kappa}-J{kappa} rearrangements have been shown in BM of µMT mice (15); and serum analysis of µMT/lpr mice revealed a markedly skewed {kappa}/{lambda} ratio with some mice having predominantly lambda serum IgG(24). Similarly, antigen-independent ongoing light chain rearrangements have been described for B cells expressing signaling incompetent receptors (51,52). It is possible that in the µMT mice, the expression of light chain can rescue the development of isotype-switched B cells as has been described for {lambda}5-deficient mice (53), providing an important role for receptor editing in forming the {gamma}H-driven repertoire. A similar contribution for receptor editing to the µH-driven repertoire has been described (49).

We cannot exclude the possibility that self-reactivity promotes the development and selection of {gamma}H-expressing cells (46), particularly in lymphopenic mice such as the µMT. In this case, ongoing rearrangements in the µMT/lpr mice should aim to select appropriate V{kappa}-J{kappa} that will provide the BCR with self-reactivity, rather than for constructing a protecting repertoire. It is also possible that light chain rearrangement occurs in the periphery at low frequency, although we could not detect RAG expression in LN or spleen of µMT/lpr mice (not shown). Recent studies have shown that lack of Fas allows a permissive environment for Igneg B cells to participate in the germinal center reaction and to acquire self-reactivity by receptor editing (54). Further selection and expansion is T-cell dependent. Similarly, we found that isotype switching in the µMT/lpr mice is T-cell independent, but subsequent selection and expansion requires the presence of T lymphocytes (14). Thus, the generation of a monoclonal autoimmune repertoire in the µMT/lpr mouse is regulated by Fas signaling but further clonal activation and expansion is T-cell dependent.

Finally, repertoire analysis of several autoimmune strains, relative to normal strains, reveals a significantly skewed IgH repertoire but a normal distribution of Ig{kappa} repertoire (41). There are also no remarkable differences between CD5+ and CD5neg in the Ig{kappa} repertoire (41). This analysis, however, was performed on IgM-expressing cells. In contrast, we obtained an oligo-monoclonal Ig{kappa} repertoire in the µMT/lpr mouse model. It is possible that in the µMT mouse, where B cell development is driven by {gamma}H receptors, the IgL chain plays an important function in determining the BCR specificity, and therefore repertoire selection in these mice reflects monoclonality of a particular V{kappa} gene. This, however, is yet to be determined.


    Acknowledgements
 
We thank Professor Gershoni and Dr Denisova from Tel Aviv University, Tel Aviv, Israel, for providing the epitope phage library and for extensive review of the manuscript. This research is supported by The Israel Science Foundation, the German–Israel Foundation for Scientific Research and Development, Young Scientists’ Program, the Colleck Research Fund and the Hirshenstrauss–Gutman Medical Research Fund.


    Abbreviations
 
AID—activation-induced cytidine deaminase

BCR—B cell receptor

BM—bone marrow

CSR—class switch recombination

ELISPOT—enzyme-linked immunosorbent spot-forming cell assay

FW3—framework 3

{gamma}H—{gamma} heavy

IL-7—interleukin 7

µH—µ heavy

Tg—transgene


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Table 1. Amino acid motifs identified for µMT/lpr serum samples in phage display epitope library
 

    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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