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The Journal of Immunology, 2006, 176: 889-898.
Copyright © 2006 by The American Association of Immunologists

Development of Functional B Cells in a Line of SCID Mice with Transgenes Coding for Anti-Double-Stranded DNA Antibody1

Gayle C. Bosma*, Jennifer Oshinsky*, Kerstin Kiefer*,{dagger}, Pamela B. Nakajima*, Deepshika Charan*, Cecil Congelton*, Marko Radic{ddagger} and Melvin J. Bosma2,*

* Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111; {dagger} Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107; and {ddagger} Department of Molecular Sciences, College of Medicine, University of Tennessee, Memphis, TN 38163


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Deletion or inactivation of anti-self (DNA) B cells has been reported in non-autoimmune mice bearing Ig transgenes that code for Abs with specificity for dsDNA or ssDNA. However, we report a case in which anti-dsDNA B cells appear to escape both deletion and inactivation. We show that B cells (B220+IgM+) can develop in non-autoimmune SCID mice bearing two site-directed transgenes, 3H9(56R) and V{kappa}8, that together code for an anti-dsDNA Ab. The B cells appear inactive, because the mice (56RV{kappa}8 SCID mice) generally lack serum Ig. However, 56RV{kappa}8 SCID mice are able to produce IgG Ab with specificity for dsDNA when they become "leaky" for T cells or are reconstituted with exogenous T cells from B cell-deficient JH–/– donors. Thus, anti-dsDNA B cells that escape deletion in 56RV{kappa}8 SCID mice appear fully functional and can differentiate, class switch, and give rise to IgG-producing cells in the presence of T cells and self-Ag.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Developing bone marrow B cells that express autoreactive Abs may, depending on the specificity and affinity of their Ab for self-Ag, be inactivated or undergo apoptosis (programmed cell death). In this way, potentially pathogenic autoimmune B cells are neutralized or deleted. This process has been illustrated using several different Ig transgene (tg)3 mouse models (1, 2, 3, 4, 5, 6). For example, Weigert and colleagues (6, 7) have shown that the deletion of anti-self B cells expressing tg-coded Ag receptors specific for dsDNA readily occurs in non-autoimmune strains of mice such as BALB/c. Some anti-dsDNA B cells may escape deletion, but these cells appear nonfunctional (8, 9, 10). B cells that express tg-coded Ag receptors with low affinity for DNA appear to be inactivated (3, 11). The actual self-Ag(s) recognized by anti-DNA B cells may be associated with membrane fragments (blebs) of dying cells (12, 13, 14) and could include determinants on DNA and chromatin as well as certain polyanions (e.g., cardiolipin and anionic phospholipids) (15, 16, 17).

Whether transgenic B cells with anti-DNA specificity are uniformly inactivated or deleted may depend not only on their affinity (or avidity) for self-Ag but also on the particular microenvironment in which these cells arise. For example, IgM+ B cells expressing an H (3H9) and an L (V{kappa}8) chain tg, which together code for an Ab with relatively low affinity for DNA (i.e., bind ssDNA but not dsDNA), appear to be partially activated when they arise in T cell-deficient mice (7). Evidence for this observation came from a comparison of the phenotype of B cells in RAG2+/– and RAG2–/– mice bearing a site-directed (sd) 3H9 tg (18) and a non-sd V{kappa}8 tg (19), designated in this paper as 3H9V{kappa}8 RAG2+/– and 3H9V{kappa}8 RAG2–/– mice. Mature peripheral B cells (CD43–IgMlowIgDhigh) were observed in both transgenic lines, but the 3H9V{kappa}8 RAG2–/– line was found to contain an additional B cell population of CD43+IgMhighIgDlow cells (7). CD43 is expressed on progenitor B and activated B cells, but not on precursor B or mature resting B cells (20, 21, 22); thus, the CD43+IgMhighIgDlow cell population was considered to represent activated B cells. However, because 3H9V{kappa}8 RAG2–/– mice are deficient in serum Ig (7), there appear to be no fully activated B cells in these mice. Full activation of 3H9V{kappa}8 B cells may require Th cells. Indeed, we recently reported that T cell reconstitution of C.B-17 SCID mice bearing the sd 3H9 and V{kappa}8 tgs (23), here denoted as 3H9V{kappa}8 SCID mice, enables the B cells in these mice to differentiate, class switch, and give rise to IgG-producing cells (24). In the absence of T cells, the B cells in 3H9V{kappa}8 SCID mice are apparently inactive, because the mice generally lack serum Ig (24). Thus, the functional status of anti-DNA B cells may depend on whether these cells arise in the presence or absence of T cells.

In this work, we report that T cell-deficient C.B-17 SCID mice bearing the sd tgs V{kappa}8 and a variant of 3H9, 3H9(56R), contain functional B cells (the variant 3H9(56R) differs from the 3H9 tg by 1 aa; i.e., it has arginine at position 56 instead of aspartate) (25). Together, the 3H9(56R) and V{kappa}8 tgs code for an Ab with relatively high affinity for dsDNA (26). How B cells escape deletion in the present case is not clear. One way that anti-DNA B cells escape deletion is by changing their receptor specificity, a process known as receptor editing (27, 28). Successful editing occurs when an autoreactive B cell makes a secondary rearrangement at one of its Ig loci, and the resulting H or L chain changes the specificity of its Ag receptor (18, 23, 25, 27, 28, 29, 30, 31). However, in SCID mice bearing 3H9(56R) and V{kappa}8 (denoted as 56RV{kappa}8 SCID mice), we would not expect receptor editing to occur at an appreciable frequency, because Ig gene rearrangement is severely impaired by the scid mutation (32). Nonetheless, we find tg-expressing B cells in the bone marrow and spleen of 56RV{kappa}8 SCID mice. Furthermore, we find that 56RV{kappa}8-expressing SCID B cells can be activated to undergo IgM-to-IgG class switching in the presence of T cells and self-Ag. We conclude that 56RV{kappa}8 scid B cells can escape both deletion and inactivation without altering their tg-coded Ag receptors.


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

BALB/c mice with the sd tgs 3H9, 3H9(56R), and V{kappa}8 (18, 23, 25) were provided by M. Weigert (Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL). These tgs were selectively crossed into C.B-17 SCID mice (C.B-17 is an Ig congenic partner strain of BALB/c). SCID mice homozygous for 3H9 or 3H9(56R) were crossed with SCID mice homozygous for V{kappa}8 to obtain 3H9/+,V{kappa}8/+ and 3H9(56R)/+,V{kappa}8/+ SCID mice, designated in this paper simply as 3H9V{kappa}8 SCID and 56RV{kappa}8 SCID mice. The 3H9(56R) and V{kappa}8 tgs were also crossed into BALB/c mice, homozygous for a disrupted RAG1 gene (RAG1–/–) (33). BALB/c RAG1–/– mice were provided by R. Hardy (Fox Chase Cancer Center). C.B-17 SCID mice bearing 3H9(56R) and a V{kappa}1 sd tg or the non-sd tg V{kappa}4 (28) were obtained in similar fashion (the V{kappa}1 sd tg was crossed into C.B-17 SCID mice from a recently constructed BALB/c V{kappa}1 transgenic line kindly provided by M. Weigert). Transgenic controls, heterozygous for the scid mutation (56RV{kappa}8 SCID/+ mice), were obtained by crossing 3H9(56R)/3H9(56R) SCID mice with V{kappa}8/V{kappa}8 C.B-17 +/+ mice. Genotyping of transgenic mice was done by PCR as described previously (18, 23). C.B-17 mice with deleted JH loci (C.B-17 JH–/– mice) (34) were provided by R. Hardy (Fox Chase Cancer Center) and used as cell donors for T cell reconstitution of 56RV{kappa}8 SCID mice. All mice were bred and maintained as specific pathogen-free mice in the Laboratory Animal Facility of the Fox Chase Cancer Center. Mice were used between 8 and 12 wk of age (unless otherwise noted) according to protocols approved by the Animal Care and Use Committee of this institution.

Flow cytometry

Suspensions of bone marrow, thymus, and spleen cells were prepared in the manner described previously (24, 35). For peripheral blood analyses, mice were bled with heparinized pipettes from the suborbital sinus. Each blood sample was added to 1.5 ml of RPMI 1640 containing 3% FCS and 25 U/ml heparin, layered onto 1.5 ml of lympholyte-M (Cedarlane Laboratories), and spun at room temperature for 20 min at 1500 x g. Cells at the interface were removed and washed three times at 4°C. Cells were stained for CD45(B220), IgM, and CD3 using allophycocyanin-conjugated anti-B220 (RA3-6B2), FITC-conjugated anti-IgM (331.1), and PE-conjugated anti-CD3 (500A2) (BD Pharmingen). Staining for the IgMa and IgMb allotypes was done using FITC-conjugated anti-IgMa (RS3.1) and biotin-conjugated anti-IgMb (AF6). Anti-IgMb staining was visualized with streptavidin-Texas Red. Analyses were performed with a FACSVantage SE flow cytometer (BD Biosciences) using FlowJo software (Tree Star). Forward and light angle scatter gates were set to exclude nonlymphoid cells. Propidium iodide staining was used to exclude dead cells.

B cell hybridomas

Spleen cells from 56RV{kappa}8 SCID mice were stimulated with LPS (50 µg/ml) for 2 days and then fused with Sp2/0 cells (36) in the manner described previously (37). Supernatants from individual hybridomas were saved for serological analysis. Total RNA from each hybridoma was obtained using TRIzol (Invitrogen Life Technologies) according to manufacturer’s instructions, and cDNA was synthesized with SuperScript II (Invitrogen Life Technologies) using 2 µg of total RNA and 1 µg of oligo(dT)12–18 primer (Invitrogen Life Technologies) in a 20-µl reaction volume. The 3H9(56R) transcript was amplified via RT-PCR with the following conditions: 50°C annealing temperature, 2.0 mM Mg+ final concentration, and 35 cycles using 1.5 U of AmpliTaq polymerase (Applied Biosystems) along with a 3H9 leader primer (5'-CTCTTCCTCCTGTCAGGAACTGCAG-3') and a CDR3 primer (3) in a 50-µl reaction volume. The V{kappa}8 transcript was amplified similarly but with the following conditions: 60°C annealing temperature, 2.0 mM Mg+ final concentration, and 35 cycles using 2.5 U of AmpliTaq polymerase (Applied Biosystems) along with the V{kappa}8 leader and V{kappa}8-J{kappa}5 oligonucleotide primers (3) in a 50-µl reaction volume. RT-PCR-amplified products were separated by electrophoresis in agarose gels (1.5%) and then gel-purified using the QIAEX II gel extraction kit (Qiagen). Clones for sequence analysis were produced using the TOPO TA cloning kit (Invitrogen Life Technologies) and underwent plasmid recovery by Perfectprep plasmid mini kit (Eppendorf). Plasmids were submitted for cycle sequencing using the ABI Prism dye terminator reaction kit and a model 377 genetic analyzer (Applied Biosystems).

Serological assays

Serum Ig({kappa}), IgM, and IgG isotype concentrations were ascertained by ELISA using purified mAbs (from BD Pharmingen) specific for Ig({kappa}), IgM, IgG1, IgG2a, and IgG2b. In some experiments, we also tested for the IgG1b allotype using monoclonal anti-IgG1b from BD Pharmingen. Assays were performed using solutions and buffers prescribed by the manufacturer. Briefly, 96-well plates (Nunc-Immuno plates) were coated with a given mAb (2 µg/ml) in coating buffer (0.01 M sodium bicarbonate and 34 mM sodium carbonate (pH 9.5)) at 4°C overnight. Plates were washed three times with PBS/0.1% Tween 20 and blocked for 1 h with PBS containing 1% BSA. Diluted sera from individual mice were then added to the wells. After a 2-h incubation, the wells were washed, and biotinylated Ab with the same isotype specificity as the Ab used to coat the wells was added along with avidin-HRP conjugate. The plates were washed 1 h, and substrate was added (tetramethylbenzidine and hydrogen peroxide). OD readings were taken at 450 nm using a KCjunior plate reader (Bio-Tek). Readings were compared with a standard curve obtained with serial dilutions of an affinity-purified monoclonal Ig of the appropriate isotype.

To assay for total IgG, we used purified goat anti-mouse IgG H+L (Southern Biotechnology Associates). The Ab was diluted in coating buffer (0.1 M sodium bicarbonate and 0.02% sodium azide (pH 9.6)) and added at 1 µg/ml to microtiter wells (Immulon 4HBX; Dynex Technologies). After overnight incubation at 4°C, the wells were washed twice with PBS/0.05% Tween 20, blocked with PBS/1% BSA/2.5 mM EDTA/0.02% sodium azide for 1 h, and washed again as described above before the addition of serial dilutions of sera from experimental and control mice. After a 2-h incubation at room temperature, the wells were washed, and goat-anti-mouse IgG chain-specific antiserum conjugate of alkaline phosphatase (Southern Biotechnology Associates) was added at a 1/1000 dilution in blocking buffer. After incubation at room temperature for 1 h, the wells were washed as described above, and paranitrophenyl phosphate (Sigma-Aldrich) was added at 1 mg/ml in alkaline phosphatase reaction buffer (0.05 M sodium bicarbonate and 10 mM Mg2Cl (pH 9.6)). OD was measured at 405 nm using an ELISA plate reader from Molecular Devices. Readings were compared with a standard curve of OD values obtained by using serial dilutions of affinity-purified mouse serum IgG (Southern Biotechnology Associates).

To assay for IgM (or IgG) anti-DNA Ab, 96-well microtiter plates (Immulon 4HBX; Dynex Technologies) were coated overnight at 4°C with a solution of 2 µg/ml avidin D (Vector Labs) in coating buffer (0.1 M sodium bicarbonate and 0.02% sodium azide (pH 9.6)). Following coating, the wells were rinsed with wash buffer (PBS/0.05% Tween 20) and blocked with blocking buffer (PBS/1% BSA/2.5 mM EDTA/0.05% Tween 20) for 1 h at room temperature. Biotinylated dsDNA was prepared as described (38), diluted in blocking buffer to 2 µg/ml, and incubated in the washed wells of the microtiter plate for 1 h at room temperature. Following incubation, unbound DNA was removed by washing with buffer; serial dilutions of sera (or hybridoma supernatant) were prepared, added to wells, and allowed to react with the bound DNA. After 2 h at room temperature, the wells were washed, and goat-anti-mouse IgM (or chain-specific goat anti-IgG) conjugated with alkaline phosphatase (Southern Biotechnology Associates) was added at a 1/1000 dilution in blocking buffer. After a 1-h incubation at room temperature, the wells were washed as described above, and paranitrophenyl phosphate (Sigma-Aldrich) was added at 1 mg/ml in alkaline phosphatase reaction buffer (0.05 M sodium bicarbonate and 10 mM Mg2Cl (pH 9.6)). OD was measured at 405 nm using an ELISA plate reader from Molecular Devices. Readings were compared with a standard IgM (or IgG) curve.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
56RV{kappa}8 SCID mice contain B cells

In an earlier study by Chen et al. (26), BALB/c mice bearing the non-sd tgs 3H9(56R) and V{kappa}8 were found to lack anti-dsDNA B cells. Thus, we were surprised to find B cells in the bone marrow and spleen of Ig-congenic BALB/c (C.B-17) SCID mice with the sd tgs 3H9(56R) and V{kappa}8 (Fig. 1). The B cells in 56RV{kappa}8 SCID mice stained weakly for IgM (Fig. 1A) and numbered ~2–3 x 106 cells per spleen (Table I) (similar results were obtained with 56RV{kappa}8 RAG–/– mice; see Fig. 1A and Table I). In contrast, 3H9V{kappa}8 SCID mice with sd tgs that code for an Ab with relatively weak affinity for DNA contained normal numbers of splenic B cells (~30 x 106 per spleen), many of which stained strongly for IgM (Table I and Fig. 1A). In 56RV{kappa}8 SCID/+ control mice, which were heterozygous for the scid mutation, most of the B cells in the bone marrow and the spleen stained weakly for IgM, although additional bright IgM-staining cells were clearly evident in the spleen (Fig. 1A). 56RV{kappa}8 SCID/+ mice contained 2- to 3-fold more splenic B cells than 56RV{kappa}8 SCID mice but 5- to 6-fold less splenic B cells than nontransgenic SCID/+ mice (Table I).



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  		FIGURE 1. Detection of B cells in bone marrow (BM) and spleen (SPL) of 8- to 12-wk-old 56RV{kappa}8 SCID (56RV{kappa}8 s/s) and 56RV{kappa}8 RAG1–/– (56RV{kappa}8 r/r) mice. Controls and experimental transgenic mice included 56RV{kappa}8 s/+, 3H9V{kappa}8 s/+, 3H9/V{kappa}8 s/s, 56RV{kappa}1 s/s, 56RV{kappa}4 s/s, 3H9V{kappa}4 s/s, and nontransgenic (non-tg) s/s and s/+ mice. Bone marrow and spleen cells from each individual were analyzed by FACS for B220 vs IgM (A and B) and IgMa vs IgMb staining of B220+-gated lymphocytes (C). The numbers above or within the boxed areas correspond to the percentage of B cells. Nonlymphoid cells were excluded from analysis by the forward and light-angle scatter gates. Several independent analyses were performed that included one or two individual mice of each genotype. The profiles shown are representative.

 

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  		Table I. Spleen cellularity and number of splenic B cells in CB17 transgenic and nontransgenic mouse lines used in this studya

 
When the 3H9(56R) chain is paired with a V{kappa}4 L chain, the resulting Ab shows higher affinity for dsDNA than the 56RV{kappa}8 Ab (26). Therefore, we asked whether C.B-17 SCID mice bearing the 3H9(56R) and V{kappa}4 tgs (56RVk4 SCID mice) would be able to develop B cells. As shown in Fig. 1B, 56RV{kappa}4 SCID mice were found to lack splenic B cells. 56RV{kappa}1 SCID mice also lacked splenic B cells, consistent with the inability of V{kappa}1 chains to veto the DNA binding specificity of the 3H9(56R) chain (25). We found that splenic B cells were also absent in 3H9V{kappa}4 SCID mice, similar to 3H9V{kappa}4 RAG2–/– mice (7). The latter finding is of particular interest, because 3H9V{kappa}4 and 56RV{kappa}8 Abs show comparable affinity for dsDNA (26). However, the actual self-Ags recognized by these two Abs in vivo are not known and may differ.

Fig. 1C shows that bone marrow and splenic B cells in 56RV{kappa}8 SCID mice expressed solely the µ-chain allotype (IgMa) of the 3H9(56R) tg (3). The IgMa staining was weak relative to that seen for B cells in the C57BL/6 x BALB/c F1 controls. The weak IgMa staining presumably reflects low abundance of cell surface IgM, and, as discussed later, this may be critical in allowing 56RV{kappa}8 B cells to escape deletion. In the 56RV{kappa}8 SCID/+ controls, we detected IgMa only in the bone marrow, but in the spleen ~25% of the splenic B cells appeared to express both IgMa and IgMb, and ~15% of the B cells showed surface IgMb only (IgMb corresponds to the µ-chain allotype coded by the wild type (wt) or non-tg allele). Note that IgMb-expressing B cells were not evident in the bone marrow or the spleen of 3H9V{kappa}8 SCID/+ mice. Cells expressing IgMb can be inferred to contain a productive rearrangement at their wt chain allele. Whether such rearrangements are Ag-induced or occur before cells encounter self-Ag is not clear and is under investigation.

B cells in 56RV{kappa}8 SCID mice can escape deletion without altering the specificity of their tg-coded Ab

We were concerned that B cells in 56RV{kappa}8 SCID mice might have escaped deletion by mutating their tgs, resulting in an Ag receptor with reduced affinity for self-Ag. In addition, VH replacement at the chain tg allele in 56RV{kappa}8 SCID mice could also potentially alter the specificity of some B cells. Therefore, to test for possible alteration of the 3H9(56R) and V{kappa}8 tgs, we obtained splenic B cell hybridomas from 56RV{kappa}8 SCID mice.

Spleen cells from two mice were separately fused with the Sp2/0 cell line (36). A total of 36 hybridomas were recovered; 14 from the first fusion (KD series) and 22 from the second fusion (KK series). All of the hybridomas produced IgM; furthermore, those in the KK series were examined for allotype, and all of them secreted IgM of the tg allotype (IgMa) (data not shown). Hybridomas in the KK series were also examined for specificity and were found to produce IgM anti-dsDNA Ab (illustrated in Fig. 2A). Hybridomas in the KD series were analyzed at the molecular level. As shown in Fig. 2, B and C, the deduced amino acid sequences of the expressed tg-coded H and L({kappa}) chain V regions corresponded (with few exceptions) to those of 3H9(56R) (25) and V{kappa}8 tgs (39). Ten hybridomas showed 3H9(56R) V regions identical with those of the original 3H9(56R) tg. One hybridoma had a cysteine for tyrosine at position 79, and three hybridomas had substitutions at positions 49, 58, 60, and 66, located near or in the CDR2 region. The latter changes involved substitution of a neutral amino acid for one with a negatively charged side group at positions that could affect binding to dsDNA (38, 40). The question of whether these and other potential substitutions in the 3H9(56R) chain actually alter Ab affinity for dsDNA is currently being examined. The L({kappa}) chain sequences were identical with the sequence of the V{kappa}8 tg except for one sequence in which phenylalanine was substituted for tyrosine at position 100 (Fig. 2C). We conclude that peripheral B cells in 56RV{kappa}8 SCID mice can escape deletion without altering their tg-coded Ab specificity.



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  		FIGURE 2. Analysis of splenic B cell hybridomas from 56RV{kappa}8 SCID mice. A, Binding curves for IgM (for four hybridomas o f the KK series) to dsDNA as a function of the concentration of IgM added to wells coated with biotinylated dsDNA. B, Deduced amino acid sequence of 3H9(56R) cDNAs from hybridomas of the KD series is shown for the region where substitutions were found. The CDR (CDR-2) is highlighted. C, Deduced amino acid sequence of V{kappa}8 cDNAs from hybridomas of the KD series is shown for the region in which one substitution was found. The CDR-3 region is highlighted. Dashed lines denote amino acids identical with those in 3H9(56R) or V{kappa}8; amino acid substitutions are indicated with bold letters.

 
Some 56RV{kappa}8 SCID mice contain serum Ig (Ig+ 56RV{kappa}8 SCID mice)

Given that 56RV{kappa}8 SCID mice contained peripheral B cells expressing unaltered tg-coded Ag receptors, it was of interest to know whether these cells were functionally inactivated. Consistent with this possibility, the B cells in 56RV{kappa}8 SCID transgenic mice showed weak staining for surface IgM, a characteristic of anergic B cells (1, 41). Furthermore, as shown in Fig. 3A, the majority of 56RV{kappa}8 SCID mice (~75%) lacked detectable serum Ig({kappa}) suggesting that these mice contained inactive B cells. However, ~25% of 56RV{kappa}8 SCID mice were found to contain Ig({kappa}) concentrations ranging from 5 to 462 µg/ml. We refer to these mice as Ig+ 56RV{kappa}8 SCID mice. In contrast to Ig+ 56RV{kappa}8 SCID mice, 56RV{kappa}8 SCID/+ mice contained relatively high concentrations of serum Ig({kappa}) ranging between 400 and 1000 µg/ml in most individuals (Fig. 3A).



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  		FIGURE 3. A, Range of serum Ig({kappa}) concentrations found in 9- to 15-wk-old 56RV{kappa}8 SCID (56RV{kappa}8 s/s) and 56RV{kappa}8 SCID/+ (56RV{kappa}8 s/+) mice. Each symbol represents a single mouse. Sixty (n = 60) of the 81 tested 56RV{kappa}8 s/s mice contained <2 µg/ml serum Ig({kappa}). B, Binding of serum IgG to dsDNA as a function of the concentration of Ig({kappa}) added to wells coated with biotinylated dsDNA. Symbols denote sera from individual Ig+ 56RV{kappa}8 SCID ({blacktriangleup}), 56RV{kappa}8 SCID/+ ({triangleup}), and nontransgenic SCID/+ ({square}) mice.

 
Three of the five Ig+ 56RV{kappa}8 SCID mice depicted in Fig. 3A with >100 µg/ml serum Ig({kappa}) and for which we had sufficient serum were tested for anti-dsDNA Ab. Serial dilutions of serum from these mice, containing from 0.1 to 2 µg/ml serum Ig({kappa}), were added to microtiter wells containing biotinylated dsDNA attached to avidin D. As indicated in Fig. 3B, IgG Ab to dsDNA was readily detectable in these mice. IgG anti-dsDNA Ab was also detectable in 56RV{kappa}8 SCID/+ mice, provided that a relatively high concentration of serum Ig was added to the wells (equivalent to ≥20 µg/ml serum Ig({kappa})). Serum Ig from age-matched C.B-17 SCID/+ mice, which lacked detectable IgG anti-dsDNA at all concentrations tested, served as a control for nonspecific binding. These results suggest that 56RV{kappa}8 SCID B cells are not functionally inactivated and, with appropriate stimulation, may differentiate into plasma cells producing IgG anti-dsDNA Ab.

Detection of T cells in Ig+ 56RV{kappa}8 SCID mice

Because SCID mice are known to be leaky for T (and B) cells (42, 43), we considered that Ig+ 56RV{kappa}8 SCID mice might have had some of their B cells activated by leaky T cells. To test for such cells, we screened peripheral blood of Ig+ 56RV{kappa}8 SCID mice for the presence of B220–CD3+ T cells. Representative results are shown in Fig. 4. CD3+ T cells, representing 3–47% of the PBLs, were detected in almost all Ig+ 56RV{kappa}8 mice (10 of 12 tested mice). With few exceptions, 56RV{kappa}8 SCID mice containing <2 µg/ml Ig({kappa}) (Ig– 56RV{kappa}8 SCID mice) lacked detectable T cells in their peripheral blood (illustrated in Fig. 4). Thus, production of serum Ig clearly correlated with the presence of leaky T cells, suggesting that these cells were responsible for the activation and differentiation of 56RV{kappa}8 SCID B cells into Ig-producing cells.



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  		FIGURE 4. Presence of serum Ig({kappa}) in 56RV{kappa}8 SCID mice correlates with the presence of leaky T cells. FACS profiles show B220 vs CD3 staining of peripheral blood lymphocytes (gated by forward and light scatter) in Ig– vs Ig+ 56RV{kappa}8 SCID mice. Numbers above boxed areas denote the percentage of T (B220–CD3+) cells.

 
Production of IgG with specificity for dsDNA in 56RV{kappa}8 SCID mice reconstituted with T cells from C.B-17 JH–/– donors

To test whether 56RV{kappa}8 SCID mice would produce the IgG anti-dsDNA Ab when reconstituted with an exogenous source of T cells, mixtures of bone marrow cells (2 x 106) and thymocytes (3 x 106) from C.B-17 JH–/– donors were injected i.v. into Ig– 56RV{kappa}8 SCID recipients. The use of donor mice genetically unable to generate B cells ensured that engrafted recipients were reconstituted with lymphocytes of the T lineage only. As illustrated in Fig. 5A, peripheral blood T cells (B220–CD3+) were detectable in 56RV{kappa}8 SCID mice injected with above cell inoculum. At 4 wk after cell transfer, 28.0 ± 13.7% of the lymphocyte-gated cells corresponded to T cells. Retesting the same mice 3 wk later showed percentages of peripheral blood T cells (75.9 ± 4.8) within the normal range (78.6 ± 4.3).



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  		FIGURE 5. Reconstitution of 56RV{kappa}8 SCID mice with T cells from C.B-17 JH–/– donors. Thymocytes (3 x 106) and bone marrow cells (2 x 106) from adult C.B-17 JH–/– mice were admixed and injected i.v. into Ig– 56RV{kappa}8 s/s recipients (8–12 wk of age). A, Percentage of T (B220–CD3+) cells in lymphocyte-gated peripheral blood of recipients at 4 and 7 wk after cell transfer. Each triangle symbol represents an individual mouse. The mean percentage (±SEM) of T cells at 4 and 7 wk after cell transfer was 28.0 ± 13.7 and 75.9 ± 4.8, respectively; the mean percentage (±SEM) of T cells in peripheral blood of young adult C.B-17 SCID/+ mice was 78.6 ± 4.3 (data not shown). B, Concentration of serum IgG in individual recipients at 2, 4, and 6 wk after cell transfer. C, Relative binding of serum IgG to dsDNA as a function of the concentration of IgG added to wells coated with biotinylated dsDNA. Each binding curve represents sera from individual recipients with ≥100 µg/ml IgG at 6 wk after cell transfer (the sera analyzed are denoted in B between the small horizontal lines).

 
Sera from 56RV{kappa}8 SCID mice reconstituted with T cells in the above manner were collected at 2, 4, and 6 wk after cell transfer and assayed for the presence of IgG. Results for one of several experiments are shown in Fig. 5B. Note that serum IgG was detectable in most recipients within 2–4 wk after cell transfer. The concentration of serum IgG varied greatly between individuals, ranging from 41 to 815 µg/ml at the sixth week after cell transfer. Two recipients failed to produce serum IgG during the 6-wk period of observation. None of the recipients produced IgG of the non-tg allotype (IgGb), and no IgG was detectable in transgenic recipients that received x-irradiated C.B-17 JH–/– donor cells (data not shown).

Sera from mice containing ≥100 µg/ml IgG 6 wk after cell transfer (in Fig. 5B) were analyzed for the presence of the IgG anti-dsDNA Ab. As shown in Fig. 5C, all six of these sera contained Ab to dsDNA. Note that the IgG anti-dsDNA binding curves were similar, suggesting that near-equivalent proportions of the IgG in each recipient corresponded to anti-dsDNA Ab. From these results, we infer that IgM+ B cells in 56RV{kappa}8 SCID mice are fully functional and can be activated by T cells and self-Ag to differentiate, class switch, and give rise to cells producing IgG anti-dsDNA Ab. It is important to note that we have not observed any evidence of autoimmune disease in T cell-reconstituted 56RV{kappa}8 SCID mice.

Production of IgG with reduced specificity for dsDNA in 56RV{kappa}8 SCID mice reconstituted with T cells derived from fetal liver of C.B-17 JH–/– donors

Activation of 56RV{kappa}8 SCID B cells by T cells from adult C.B-17 JH–/– mice could be considered the consequence of a graft-vs-host reaction. The T cells in this case are derived from a B cell-deficient mouse and may recognize 56RV{kappa}8 B cells as foreign. Alternatively, activation could involve recognition by naive T cells of DNA associated self-Ag presented by MHC class II molecules on the surface of 56RV{kappa}8 SCID B cells. In an attempt to achieve T cell tolerance to 56RV{kappa}8 SCID B cells, we transferred C.B-17 JH–/– fetal liver cells into neonatal 56RV{kappa}8 SCID recipients to allow for concurrent development of donor T and host B cells in the same microenvironment.

Results for the cell transfer of C.B-17 JH–/– fetal liver cells to neonatal 56RV{kappa}8 recipients are shown in Fig. 6. Each of eight recipients was injected i.p. with 107 cells; control recipients were injected with cell suspension buffer only (represented by {triangleup}). Most of the test recipients (represented by {blacktriangleup}) showed ≥50% T cells in their peripheral blood by 7 wk of age (Fig. 6A), although IgG was not detectable in recipients before 12 wk of age (Fig. 6B and data not shown). At 15 wk of age, all but one recipient produced IgG, and at 23 wk of age, all recipients contained ≥100 µg/ml IgG and showed normal percentages of T cells (Fig. 6B). However, appreciable levels of IgG anti-dsDNA Ab (absorbance, >1) were present in only three of the eight recipients (Fig. 6C). As indicated in Fig. 7, the two recipients with the highest levels of Ab (mouse no. 17 and mouse no. 22) showed similar IgG anti-dsDNA binding curves. Note that the binding curves for mouse number 17 at two different time points (15 and 23 wk) are superimposable; only one time point is shown for mouse number 22, because this mouse did not contain detectable IgG at the 15-wk interval. Dramatically reduced binding of IgG to dsDNA was observed in the remaining recipients (see Fig. 7), suggesting that these mice may have produced IgG Abs coded by mutated tgs. This possibility remains to be tested.



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  		FIGURE 6. Reconstitution of 56RV{kappa}8 SCID mice (starting at birth) with T cells derived from fetal liver of C.B-17 JH–/– donors. Fetal liver cells (107) from 17-day-old embryos were injected i.p. into newborn (2-day-old) 56RV{kappa}8 SCID recipients. Control 56RV{kappa}8 SCID recipients were injected with cell suspension (RPMI 1640) buffer only. Test and control recipients are individually denoted with {blacktriangleup} and {triangleup}, respectively. A, Percentage of T (B220–CD3+) cells in peripheral blood of recipients at 7 and 24 wk after cell transfer. B, Concentration of serum IgG in individual recipients at 8, 15, and 23 wk after cell transfer. C, Relative binding of serum IgG to dsDNA at 8, 15, and 23 wk after cell transfer.

 


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  		FIGURE 7. Binding of serum IgG to dsDNA as a function of the concentration of IgG added to wells coated with biotinylated dsDNA. Sera were from the recipients described in Fig. 6 and taken at 15 and 23 wk after cell transfer (denoted with {blacksquare} and {triangleup}, respectively). Results for the two recipients with the highest levels of IgG anti-dsDNA (nos. 17 and 22) are shown in two panels on the left; results for the remaining six recipients (nos. 14, 15, 16, 18, 19, and 21) are shown in the two panels on the right.

 
We conclude from Figs. 6 and 7 that reconstitution of 56RV{kappa}8 SCID mice with T cells derived from fetal liver of C.B-17 JH–/– donors starting at birth does not result in efficient activation of 56RV{kappa}8-expressing B cells. Activation may generally fail to occur, because most developing donor T cells are made tolerant to 56RV{kappa}8-expressing B cells and the self-Ags recognized by these Ag-presenting B cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The detection of functional B cells with specificity for dsDNA in 56RV{kappa}8 SCID mice was surprising for two reasons. First, B cells with specificity for dsDNA were not previously detected in BALB/c mice bearing the non-sd tgs 3H9(56R) and V{kappa}8 (26); i.e., splenic B cell hybridomas from these mice did not produce Abs that bound dsDNA. In fact, all but one of 27 such hybridomas from these mice showed a deletion of the 3H9(56R) tg array and expressed endogenous VH genes (26). Second, whereas B cells with anti-dsDNA specificity have been detected previously in non-autoimmune BALB/c mice bearing the non-sd tg, 3H9, these B cells (3H9/V{lambda}1 B cells) were reported to be anergic (8). Transgenic anti-dsDNA B cells have also been detected in BALB/c RAG2–/– mice bearing the non-sd tgs 3H9 and V{lambda}2 (9, 10). Although these B cells (3H9/V{lambda}2 B cells) proliferated and showed up-regulated expression of B7.2 and surface IgM in response to IgM cross-linking in the presence of CD40L-CD8 and IL-4, they did not secrete IgM Ab (10). In contrast to these earlier findings with non-sd tgs, we have found that splenic B cell hybridomas from 56RV{kappa}8 SCID mice with the sd tgs 3H9(56R) and V{kappa}8 produce IgM anti-dsDNA Ab. Moreover, the B cells in these mice differentiate into IgG anti-dsDNA-producing cells in the presence of leaky T cells or exogenous T cells from C.B-17 JH–/– donors. As discussed below, we conclude that some 56RV{kappa}8-expressing SCID B cells must escape both deletion and inactivation.

How do B cells escape deletion in 56RV{kappa}8 SCID mice?

Although we would expect receptor editing to be severely impaired by the scid defect, we initially thought that somatic mutation of the tgs or even a low frequency of successful editing among developing 56RV{kappa}8 SCID B cells may have enabled some cells to change their receptor specificity and escape deletion in the bone marrow. To test for these possibilities, we obtained IgM-producing splenic B cell hybridomas from 56RV{kappa}8 SCID mice. We found these hybridomas to produce IgM of the tg allotype (IgMa) with specificity for dsDNA. Moreover, the deduced amino acid sequences of tg-coded H and L({kappa}) chain variable regions were found to be identical with those of 3H9(56R) and V{kappa}8 in the majority of the hybridomas analyzed. Furthermore, no VDJH or VJ({kappa}) rearrangements have been detected to date at the wt (non-tg) alleles, although some hybridomas have been found to contain D-to-J rearrangements (data not shown). We conclude that most splenic IgM+ B cells in 56RV{kappa}8 SCID mice express unaltered 3H9(56R) and V{kappa}8 tgs.

One may question whether the form or abundance of DNA-associated self-Ag(s) differs significantly in 56RV{kappa}8 SCID mice from that in wild-type mice. The DNA protein kinase deficiency in SCID mice could potentially affect the composition, antigenicity, or availability of nuclear self-Ags in apoptotic bodies of dying cells such as, for example, DNA associated protein complexes involved in the repair of dsDNA breaks (12). If so, arising B cells in 56RV{kappa}8 SCID mice might not be efficiently deleted due to an altered form or abundance of self-Ag. However, such an alteration in self-Ag would not be expected in 56RV{kappa}8 RAG1–/– mice. These mice are not deficient for DNA protein kinase activity, and yet they showed the same B cell phenotype as 56RV{kappa}8 SCID mice (see Fig. 1A and Table I). Thus, B cell escape from deletion in 56RV{kappa}8 SCID mice cannot be attributed to a "missing" self-Ag resulting from the scid mutation or to some other unique property of the SCID microenvironment.

How then do 56RV{kappa}8-expressing IgM+ B cells escape deletion in SCID mice? We suggest that they may do so by down-regulating (or not up-regulating) their expression of surface IgM in the presence of self-Ag. Precedent for this suggestion comes from earlier reports of low surface IgM on tolerant (nondeleted) anti-self B cells in the hen-egg-lysozyme (HEL) transgenic model of Goodnow et al. (1, 44). Nondeleted B cells in the 56RV{kappa}8 SCID model also show a low abundance of surface receptor as evidenced by low or dull staining for IgM (Fig. 1). A low abundance of receptor on the cell surface could result in poor Ag receptor cross-linking by self-Ag and inadequate signaling of the B cell to initiate receptor editing or apoptosis. Down-regulation of receptor expression would seem to be an effective means of escape from deletion in 56RV{kappa}8 mice. This assumption can be inferred from the relatively small difference in number of splenic B cells between 56RV{kappa}8 SCID and 56RV{kappa}8 SCID/+ mice. The latter mice are able to edit their Ag receptor, and yet they were found to contain only 2- to 3-fold more splenic B cells than 56RV{kappa}8 SCID mice (Table I).

Other factors may also be key to the survival of anti-dsDNA B cells in 56RV{kappa}8 SCID mice. For example, there is evidence in the HEL transgenic model that anti-self B cells show premature death due to their exclusion from follicular sites by other competitor B cells (45, 46). Thus, in 56RV{kappa}8 SCID mice, it might be argued that the low numbers of anti-dsDNA B cells is simply due to the enhanced survival of these cells in the absence of other competitor B cells. If this argument were to suffice, however, we would also expect low numbers of anti-dsDNA B cells in 56RV{kappa}1 and 56RV{kappa}4 and 3H9V{kappa}4 SCID mice. But no B cells were detectable in these mice (Fig. 1B). Clearly, additional variables such as the specificity and affinity of tg-coded Abs for self-Ag in vivo must also be important. A comparison of the B cell phenotype in 56RV{kappa}8 SCID and 3H9V{kappa}4 SCID mice illustrates this point. 3H9V{kappa}4 SCID mice failed to develop B cells even though the affinity of their tg-coded Ab for dsDNA (and ssDNA) is comparable to that of the 56RV{kappa}8 Ab (26). This finding suggests that the actual DNA-associated self-Ag(s) recognized by these two Abs in vivo may differ. No deletion of B cells was evident in 3H9V{kappa}8 SCID mice, because they contained normal numbers of splenic B cells (Table I). This result is consistent with the much weaker binding of the 3H9V{kappa}8 vs 56RV{kappa}8 Ab to DNA (26). The 3H9V{kappa}8 Ab may also show weaker binding to phospholipids than the 56RV{kappa}8 Ab (15). In point of fact, single-chain variable fragments of 3H9(56R) bind ~4-fold better to phosphatidylserine than single-chain fragments of 3H9 (17). Phosphatidylserine is an Ag expressed on the surface of apoptotic cells. Thus, differing development fates of B cells in 3H9V{kappa}8 and 56RV{kappa}8 SCID mice could reflect differences in Ag receptor binding to the surface of apoptotic cells.

Although anti-dsDNA B cells were found to be present in 56RV{kappa}8 SCID mice, no such B cells were evident in BALB/c wt mice bearing non-sd 3H9(56R) and V{kappa}8 tgs (56RV{kappa}8 wt mice), as reported earlier by Chen et al. (26). If the survival of anti-dsDNA B cells in the presence of other competitor B cells is severely compromised, similarly as for anti-self B cells in the HEL transgenic model (45, 46), this could help explain the difference in phenotype between the 56RV{kappa}8 wt mice (with competitor B cells) and 56RV{kappa}8 SCID mice (without competitor B cells). The difference in phenotype might also relate in part to differences in tg location and copy number. The non-sd tgs 3H9(56R) and V{kappa}8 were in the form of VDJH-Cµ and VJ-C{kappa} constructs, each integrated into the genome at an unknown site and, in the case of 3H9(56R), in multiple copies (19, 26). In mice with the sd version of these tgs, as in 56RV{kappa}8 SCID mice, the J regions of one H and one L({kappa}) allele have been replaced with a single VDJH and VJ({kappa}) coding segment of the 3H9(56R) and V{kappa}8, respectively (23, 25). Given these differences, the timing and extent of tg expression in the developing B cells of mice with the sd vs non-sd tgs could differ significantly, such that deletion of 56RV{kappa}8-expressing B cells is more efficient in the 56RV{kappa}8 non-sd tg mice than 56RV{kappa}8 SCID mice.

T cell-dependent activation of 56RV{kappa}8 SCID B cells

Evidence for T cell-dependent activation of anti-dsDNA B cells in non-autoimmune transgenic mice has been reported previously. Erikson and colleagues (47) found that BALB/c mice with both the non-sd tg 3H9 and a tg coding for influenza hemagglutinin produced serum 3H9/{lambda} Ab-specific for dsDNA when engrafted with transgenic T cells that recognize the hemagglutinin Ag. T cell-dependent production of an anti-dsDNA Ab has also been reported in non-autoimmune C57BL/6 mice bearing the 3H9 or 3H9(56R) sd-tgs (48, 49). Engraftment of these mice with T cells from allogeneic C57BL/6 (bm12) donors to induce a chronic graft-vs-host reaction resulted in markedly elevated serum levels of anti-dsDNA Abs. Possibly, a mild chronic graft-vs-host reaction is induced in 56RV{kappa}8 SCID mice upon reconstitution with T cells from adult, B cell-deficient C.B-17 JH–/– mice, and this reaction results in activation of anti-dsDNA B cells. However, we think is it is more likely that activation of anti-dsDNA B cells (particularly in 56RV{kappa}8 SCID mice with leaky T cells) involves naive T cell recognition of DNA associated self-Ag presented by MHC class II molecules on the surface of 56RV{kappa}8 SCID B cells.

Given previous reports (3, 5, 6, 7, 8, 26, 47) that transgenic B cells with anti-dsDNA specificity are either deleted or inactivated in non-autoimmune mouse strains, we expected that anti-dsDNA B cells in 56RV{kappa}8 SCID mice would be inactivated. Consistent with this expectation, most 56RV{kappa}8 SCID mice lacked detectable serum Ig, and their B cells stained weakly for IgM, a phenotype associated with inactivated B cells (1, 41). However, if anti-dsDNA B cells in 56RV{kappa}8 SCID mice were really inactivated, as opposed to just inactive, we would not expect these cells to be activated by T cells in the presence of self-Ag. Indeed, Goodnow and colleagues (50) showed in the HEL transgenic model that anti-self B cells could not be activated by collaborating T cells in the presence of tolerizing self-Ag (lysozyme). In fact, when mice with anergized HEL-specific B cells were provided HEL-specific CD4+ T cells in the presence of self-Ag, the B cells appeared to be eliminated rather than activated (51). In contrast, in 56RV{kappa}8 SCID mice, anti-self B cells were activated and gave rise to cells producing IgG anti-dsDNA Ab when these mice developed leaky T cells or were reconstituted with T cells from adult C.B-17 JH–/– donors. We conclude that dsDNA B cells that escape deletion in 56RV{kappa}8 SCID mice are fully functional. Although we reported evidence for fully functional anti-DNA B cells in 3H9V{kappa}8 SCID mice (24), these B cells code for an Ag receptor with relatively low affinity for DNA (26), and there is no evidence of B cell deletion in these mice (see Table I).

Regulation of anti-dsDNA production in T cell-reconstituted 56RV{kappa}8 SCID mice

Following up on their earlier work (47), Erikson and colleagues (52) recently reported that CD4+CD25+ T regulatory cells (Treg cells) (53, 54, 55) can inhibit T cell-dependent production of anti-DNA/chromatin Ab. The following question thus arises: are Treg cells responsible for the low level of anti-dsDNA Ab in the T cell reconstituted 56RV{kappa}8 SCID mice depicted in Figs. 6 and 7? In these mice, reconstitution is initiated immediately after birth such that donor T cells develop in the presence of transgenic B cells. Under these conditions, one might propose that Treg cells arise and inhibit activation of 56RV{kappa}8-expressing B cells. Thus, in an initial attempt to inhibit possible silencing of 56RV{kappa}8 B cells by Treg cells, we injected recipient nos. 18, 19, and 21 in Fig. 7 with 200 µg/ml anti-CD25 mAb (PC61) (56) twice weekly for 3 wk. This experiment did not result in any increase in anti-dsDNA Abs (results not shown). Other experiments and analyses are planned to test for possible Treg cell inhibition of anti-dsDNA production in the above cell transfer system. Nonetheless, as suggested in Results, we propose another explanation for the low level of anti-dsDNA Ab. Specifically, we hypothesize that most arising T cells in the above cell transfer system are rendered tolerant to self-Ag presented by MHC class II molecules on the surface of 56RV{kappa}8 SCID B cells and that this outcome results in markedly reduced B cell activation and little or no anti-dsDNA Ab. We suggest that lack of anti-DNA Ab in 56RVk8 SCID/+ (and 3H9Vk8 wt) mice could also reflect a similar failure to activate anti-DNA B cells by T cells.

In sum, we have shown that some anti-dsDNA B cells appear to escape both deletion and inactivation in 56RV{kappa}8 SCID mice. We believe that these mice (and 56RV{kappa}8 RAG1–/– mice as well) may prove valuable in ascertaining how anti-dsDNA B cells escape deletion in the absence of receptor editing. Also, because these cells appear fully functional when they develop in the absence of T cells, the model should allow one to investigate the potential role of T cells in B cell tolerance to DNA-associated self-Ags.


   Acknowledgments
 
The assistance of the following core facilities of the Fox Chase Cancer Center is gratefully acknowledged: the Flow Cytometry and Cell Sorting Facility, Laboratory Animal Resources, and the Hybridoma Facility. We thank M. Weigert for providing the original BALB/c mice with the 3H9, 3H9(56R), V{kappa}1, V{kappa}4, and V{kappa}8 tgs, and R. Hardy for C.B-17 JH–/– mice. We also thank R. Hardy and K. Campbell for discussion and review of the manuscript, and R. Brooks and K. Trush for help in formatting the text and figures.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 work was supported National Institutes of Health Grants CA06927 and CA04946 and an appropriation from the Commonwealth of Pennsylvania. Back

2 Address correspondence and reprint requests to Dr. Melvin J. Bosma, Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111. E-mail address: melvin.bosma{at}fccc.edu Back

3 Abbreviations used in this paper: tg, transgene; sd, site directed; HEL, hen egg lysozyme; Treg, T regulatory cell; wt, wild type. Back

Received for publication September 2, 2005. Accepted for publication October 24, 2005.


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