Vh family utilization by igg anti-dnasecreting lymphocytes derived from autoimmune mrl-lprlpr mice.
код для вставкиСкачать56 1 VH FAMILY UTILIZATION BY IgG ANTI-DNA-SECRETING LYMPHOCYTES DERIVED FROM AUTOIMMUNE MRL-ZprlEpr MICE DENNIS M. KLINMAN, DALE K. DELLACQUA, JACQUELINE CONOVER, and KONRAD HUPPI Objective. To evaluate the heterogeneity of the IgG anti-DNA autoantibody response of MRLlZpr mice. Methods. B cell clones were grown on nitrocellulose membranes. Those producing IgG anti-DNA antibodies were identified by a modified enzyme-linked immunospot assay, and their utilization of IgVH genes was determined by hybridization. Results. Four hundred sixty-eight IgG anti-DNAsecreting colonies were derived from 9 autoimmune MRLlZpr mice. Individual VH families contributed to anti-DNA production at a frequency roughly proportional to their representation in the expressed repertoire (except for the more frequent use of VH 7183 and less frequent use of VH 36-60). In individual mice, anti-DNA antibodies were encoded by 2-5 different IgVH families, with no single family constituting more than 2 7 4 5 % of any animal's anti-DNA response. Conclusion. The IgG anti-DNA response of individual MRLlZpr mice is oligoclonal. From the Laboratory of Retrovirus Research, Center for Biologics Evaluation and Research, Food and Drug Administration, and the Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Dennis M. Klinman, MD, PhD: Laboratory of Retrovirus Research, Center for Biologics Evaluation and Research, Food and Drug Administration; Dale K. Dellacqua, BS: Laboratory of Retrovirus Research, Center for Biologics Evaluation and Research, Food and Drug Administration; Jacqueline Conover, BS: Laboratory of Retrovirus Research, Center for Biologics Evaluation and Research, Food and Drug Administration; Konrad Huppi, PhD: Laboratory of Genetics, National Cancer Institute, National Institutes of Health. Address reprint requests to Dennis M. Klinman, MD, PhD, Laboratory of Retrovirus Research, Center for Biologics Evaluation and Research, Food and Drug Administration, Building 29A, Room 3 D 10, Bethesda, MD 20892. Submitted for publication September 22, 1992; accepted in revised form November 10, 1992. Arthritis and Rheumatism, Vol. 36, No. 4 (April 1993) Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the overproduction of pathogenic autoantibodies. Mice of the MRLllpr strain provide a useful model for studying the pathogenesis of this disease. These mice develop an autoimmune-mediated glomerulonephritis and arthritis similar to that seen in humans with lupus (1,2). They also express a number of immune abnormalities, notably the accumulation of atypical T lymphocytes and the hyperactivation of B lymphocytes (3-9). Increased production of IgG anti-DNA antibodies, a hallmark of human SLE, also occurs in MRLllpr mice (10,ll). Autoantibodies of this specificity form immune complexes that contribute to the development of life-threatening glomerulonephritis (12,13). The concentration of IgG anti-DNA antibodies at sites of tissue injury correlates with disease activity (14-17). Although B cell secretion of anti-DNA is critical to disease pathogenesis, the clonal diversity of the B cells producing these autoantibodies remains uncertain (3,18; for review, see ref. 19). In an attempt to resolve this issue, investigators have studied and sequenced the immunoglobulin heavy chain variable regions (IgVH) utilized by anti-DNA-secreting B cells (20-26). Early work by Shlomchik et a1 evaluated the anti-DNA response in 5 MRLllpr mice, and showed that individual animals used no more than 2 different VH families to encode IgG anti-DNA antibodies (21,27). Based on these data, it was concluded that the anti-DNA response of individual mice was monoclonal or pauci-clonal. By comparison, Tillman et a1 studied 9 New Zealand black x New Zealand white (NZB X NZW) mice and found that individual animals utilized up to 10 different clones and 4 different VH families to encode IgG anti-DNA antibodies, leading those inves- 562 KLINMAN ET AL tigators t o conclude that the anti-DNA response was oligoclonal (25). Virtually all of the investigations in this field have relied upon hybridomas as their source of autoantibody-secreting cells (21-26,28). Yet recent evidence suggests that a large fraction of lymphocytes secreting IgG autoantibodies in vivo may not produce stable hybrids, and that hybridoma panels may therefore be selectively skewed toward the expression of a nonrepresentative subpopulation of B cells (29,30). Thus, an alternative cloning strategy was devised t o study a more diverse and potentially representative set of anti-DNA-secreting B cells. In the present study, colonies of MRLllpr mouse B cells were grown in short-term culture (31,32), and their IgVH family utilization was examined. This approach yielded large numbers of antiDNA-secreting colonies per animal and was free of the selection pressures imposed b y hybridization and long-term culture. Previous reports have shown that the IgG antibody-secreting clones identified by this strategy were representative of B cells that had been secreting Ig under physiologic conditions in vivo (33). Our results indicate that 1) within the MRLllpr strain as a whole, all VH families examined were able t o encode anti-DNA antibodies, 2) within individual mice, 2-5 different V H families were used t o produce IgG anti-DNA antibodies, and 3) individual mice relied on a single VH family to encode only 27-65%, a t maximum, of their total anti-DNA response. MATERIALS AND METHODS Animals. MRL-lpr/lpr (MRWlpr) mice were obtained from Jackson Laboratories (Bar Harbor, ME). All mice used in these experiments were 6-month-old females and had active glomerulonephritis (documented by >2+ proteinuria). Colony culture system. The method for growing B cell colonies from MRLllpr mice was adapted from that previously described by Kelsoe (31,32). Single cell suspensions were made from the spleens of freshly killed animals in medium consisting of RPMI 1640 supplemented with 10% fetal calf serum (FCS), 5 x lOP5M2-mercaptoethanol, 2 mM L-glutamine, 10 mM HEPES buffer, 0.11 mg/ml sodium pyruvate, and penicillin (50 unitdml) and streptomycin 0.11 pghl(34). Cells were washed twice and centrifuged over a discontinuous density gradient. Lymphocytes were isolated from the 50--60% Percoll fraction and cultured on sterile Immobilon-P discs (Millipore, Bedford, MA) at a concentration of 5 x lo4 cells/ml in complete medium supplemented with 15 &ml lipopolysaccharide (LPS) (33). Preliminary experiments showed that colony formation was maximal following 6 days of culture at 37°C in a 5% C02-95% air incubator. Enzyme-linked immunospot assay. Flat-bottom Immulon I microtiter plates (Dynatech, Alexandria, VA) were coated with goat anti-mouse Ig (Cappel, Cochranville, PA) or salmon sperm DNA (Sigma, St. Louis, MO) (after pretreatment with mouse bovine gamma globulin) and then blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) as previously described (35). Serial dilutions of single cell suspensions, starting with lo6 cells/ well, were incubated on antigen-coated plates for 7 hours at 37°C in a 5% C02-95% air incubator (34). The cells were washed away with PBS/O.OS% Tween 20 and the plates overlaid for 2 hours with phosphatase-conjugated antibodies to mouse IgM or IgG (Kirkegaard and Perry, Gaithersburg, MD). The antibodies produced by individual B cells that bound to the plate were visualized by addition of a 5-bromo3-chloroindolyl phosphate solution (Sigma) in a low-melt agarose kept at 50°C; phosphatase acts on this substrate to produce a blue spot that cannot diffuse through the agarose once it solidifies at room temperature (36). The dilution of cells producing 20-40 spots per well was used to determine the total number of antibody-specific B cells per sample. The sensitivity and specificity of this assay has been documented in antigen-inhibition tests and in studies involving antigenspecific hybridoma cell lines (34,36,37). Detection of Ig-secreting colonies. Immobilon P membranes were coated with salmon sperm DNA (Sigma) or goat anti-mouse Ig (Kirkegaard and Perry), at a concentration of 10 pg/ml in borate buffer, pH 8.4, and then blocked with 2% BSA plus 1% gelatin in PBS (34,38). Culture discs containing actively growing B cell colonies were washed twice with complete medium and then overlaid with an antigen-coated membrane. Following a 6-hour incubation the membrane was removed, washed extensively with PBS-0.5% Tween 20, and treated with phosphatase-conjugated antibodies to mouse IgM or IgG (Southern Biotechnology, Birmingham, AL). Antibodies secreted by the B cell colonies that bound to the antigen-coated membrane were visualized by addition of BCIP (Sigma). Colony hybridization assay. Discs containing B cell colonies were washed twice in PBS and then treated with a fresh solution of 1% buffered neutral formalin (Sigma) in PBS for 7 minutes at room temperature. Discs were washed 3 times in PBS-Tween 20, immediately prehybridized, and then hybridized overnight to a nick-translated probe of specific activity 1.42.0 X 10' counts per minute/pg in 50% formamide at 42°C (39). After washing in 0 . 1 % ~SSC ( I x SSC = O.15M NaCl and O.015M sodium citrate) for 15-60 minutes at 65°C the discs were exposed to Kodak AR-2 film (Eastman-Kodak, Rochester, NY) at -70°C in the presence of an intensifying screen. Probes. DNA probes used in this study have been reported previously (39) and correspond to VH gene families as follows: X24 is a 500-basepair Eco RI insert, 7183 is a 900-bp Sac I insert containing the entire VH coding region, J558 is a 700-bp Bum HIIBst EII fragment, 5606 is a 350-bp Hind IIISac I fragment, S107 is a 250-bp Hinf IIBst EII fragment, 36-60 is a 2,400-bp Hind I11 fragment, and 3609 is a 2000 bp Eco RYBarn HI fragment. All VH fragments were subcloned into pGEM3Z. The derivation of these probes and their specificity under the hybridization conditions used in this investigation have been previously described (39). 563 VH FAMILY USAGE BY ANTI-DNA-PRODUCING CELLS Analysis of VH families used by IgG anti-DNAsecreting colonies. Discs containing B cell colonies were first analyzed for Ig production by the colorimetric assay described above (to detect IgG anti-DNA-secreting cells) and then fixed with formalin and hybridized to VH-specific probes. Results from these 2 analyses were integrated by juxtaposing antigen-coatedmembranes (which identified IgG anti-DNA-secreting colonies) over autoradiographs (identifying colonies utilizing specific V H families) through use of alignment markings placed on all membranes and discs (Figure 1). Table 1. Repertoire comparison of in vivo-activated cells versus in vitro-stimulated colonies* IgM % IgM- or IgG-secreting cells/colonies In vivo-activated B cellst LPS-induced B cell colonies$ % DNA-specific Ig-secreting cellslcolonies5 In vivo-activated B cellst B cell colonies$ 34.6 52.7 ~ 65.4 t 11.3 47.3 t 4.5 1.9 t 0.7 2.8 t 0.9 - * Values are Isotype of Ig secreted by B cell colonies derived from MRLllpr mice. Preliminary experiments were conducted to establish conditions that would maximize the formation of Ig-secreting colonies from MRL/ lpr mice. Minor variations on the method developed by Kelsoe (31,32) proved optimal. For experiments reported herein, splenic lymphocytes were plated at a concentration of 5 X lo4 cellslml in medium supplemented with 10% FCS and 15 pg/ml LPS. This resulted in the formation of discrete Ig-secreting colo- 1) GROW COLONIES IN VlTRO CULTURE DISC 2) OVERLAY WITH DNA-COATED MEMBRANE TO IDENTIFY COLONIES PRODUCING IgG ANTI-DNA ANTIBODIES 8.3 4.5 1.8 2 0.6 1.6 2 0.5 ~~~ RESULTS 5 ? IgG 3) FIX AND HYBRIDIZEWITH lgVH GENE SPECIFIC PROBE HYBRIDIZED CULTURE DISC DNA-COATED MEMBRANE I 4) OVERLAY HYBRIDIZEDCULTURE DISC AND DNA-COATED MEMBRANE TO REVEAL lgVH GENES USED BY DNA SPECIFIC CLONES OVERLAY RESULTS Figure 1. Technique to identify IgVH genes used by IgG antiDNA-secreting colonies. 0 = B cell colony; * = alignment spot; 0 = IgVH positive; 0 = DNA positive; @ = DNA/IgVH positive. the mean 2 SD percent, from assays performed on cells from at least 6 mice per group. LPS = lipopolysaccharide. t Freshly isolated splenic B cells from unmanipulated mice were analyzed using the enzyme-linked immunospot assay (34). $ Colonies were grown on Immobilon P membranes for 6 days, as described in Materials and Methods. § % DNA-specific cells was calculated by the formula (no. of anti-DNA-secreting cells/total no. of Ig-secreting cells) x 100%. nies by -2.5% of the original B cell inoculum after 6 days of culture. This is similar to the frequency of B cells actively secreting Ig in vivo in MRLllpr mice of this age (range 1.4-2.9% [331). The isotype of Ig produced by these colonies was determined by overlaying the culture discs with Immobilon P membranes that had been precoated with goat anti-mouse Ig. Antibodies secreted by B cell colonies bound to these anti-Ig-coated membranes and were detected in an isotype-specific colorimetric assay (34,36,37). This strategy identified both the number and the location of Ig-secreting colonies. As seen in Table 1, slightly fewer than one-half of M R L / fpr colonies produced IgG. This finding is consistent with previous reports showing that B cells from adult MRLllpr mice frequently produce IgG in vivo (40) and continue to produce that isotype at a modestly lower frequency when stimulated with LPS in vitro (Table 1 and ref. 33). Additional experiments revealed that the proportion of colonies secreting IgG antibodies reactive with DNA was similar to the fraction of B cells producing those antibodies in vivo (Table 1). IgVH families used by B cell colonies derived from MRLlZpr mice. Kelsoe previously showed that treating B cell colonies with formalin would fix the colonies to the culture disc and permeabilize their membranes such that cellular messenger RNA could be detected by conventional gene hybridization techniques (31,32). In the present study, B cell colonies grown for 6 days were treated with formalin and the culture discs then hybridized with VH farnily-specific probes. To ensure a high degree of specificity with KLINMAN ET AL 564 these probes, high-stringency wash conditions were rigorously maintained (39). Results from these studies are shown in Table 2. Of the 1,345 colonies examined, a majority utilized 5558, followed, in order, by 7183, 36-60, 5606, S107, 3609, and X24. Analysis of IgG anti-DNA-secreting colonies from MRLlZpr mice. To identify B cell colonies secreting IgG anti-DNA antibodies, culture discs were overlaid with DNA-coated membranes. From 0.3% to 4.8% (mean 1.9%) of the IgG-secreting colonies from 6-month-old MRLllpr mice secreted antibodies that bound to these DNA-coated membranes. This is similar to the previously reported fraction of in vivoactivated B cells producing IgG anti-DNA antibodies in animals of this strain (0.6-7.7%, mean 2.8%) (33). The discs were then treated with formalin and probed to determine the number and location of colonies utilizing specific IgVH families. As diagrammed in Figure 1, data from these 2 assays could be integrated by superimposing the DNA-coated membranes (identifying anti-DNA-secreting colonies) with autoradiographs (identifying colonies utilizing specific IgVH families). This procedure, accomplished with the aid of preestablished alignment spots, allowed us to determine which VH gene families were used to encode anti-DNA antibodies. Four hundred sixty-eight IgG anti-DNAsecreting colonies from 9 MRLllpr mice were studied. All available probes were used in the analysis of 6 of these animals; for technical reasons, the other 3 mice were studied using only 3-5 probes. VH family utilization was analyzed independently in each animal. Results were then combined to assess the heterogeneity of the anti-DNA response in this strain as a whole. Table 2. mice* IgVH gene utilization by B cell colonies from MRLllpr % IgVH-positive colonies Probe (n = 1,345) Total IgG anti-DNA (n = 468) 7183 J558 3606 36-60 X24 S107 3609 15.7 t 8.4 51.5 -C_ 11.8 9.3 2 5.9 11.4 2 6.8 2.3 2 1.2 6.1 2 4.4 3.7 2 3.8 29.2 2 9.9 47.2 2 19.1 8.6 2 8.1 5.2 2 6.3 2.8 2 5.3 5.0 t 2.8 2.0 2 1.1 * B cell colonies were hybridized with IgVH-specific probes. The number of colonies using each VH family was divided by the total number of hybridizing colonies on that filter to calculate percent VH utilization. Values are the mean 2 SD of the data from all animals studied with a particular probe (n = 7-9 animals for each probe). Table 3. Utilization of IgVH families by IgG anti-DNA-secreting colonies in 6 MRLllpr mice* % IgVH-positive colonies Mouse Probe 7183 Probe J558 Probe 5606 Probe 36-60 Probe X24 Probe S107 Probe 3609 1 2 3 4 5 6 26.7 30.0 35.7 11.1 11.1 33.3 20.0 22.7 21.2 52.9 65.4 57.7 20.8 0 15.0 0 14.3 4.1 14.3 0 3.8 0 0 0 0 14.3 0 0 0 0 0 0 7.9 0 0 0 0 0 0 0 11.1 5.9 * Values are the percent of I& anti-DNA-secreting colonies from 6 MRLllpr mice hybridizing with this set of VH family-specific probes. These animals represent the subset of mice presented in Table 2 for which data using every probe were available. It should be noted that up to 36% of the colonies from individual mice did not hybridize with any of the probes examined; these presumably represented colonies that utilized an IgVH region for which no probe was available. As seen in Table 2, 1) all VH gene families examined were able to encode anti-DNA antibodies, and 2) most VH families contributed to the production of IgG anti-DNA autoantibodies at a frequency roughly proportional to their representation in the expressed repertoire, although 3) there was a disproportionate increase in the use of VH 7183 (P < 0.001, by chi-square test) and decrease in the use of VH 36-60 (P < 0.001) by colonies secreting anti-DNA antibodies in MRL/lpr mice. We then examined the frequency with which VH gene families were used to produce IgG anti-DNA antibodies in individual MRLllpr mice. Only those animals examined with all 7 probes were included in this analysis. As seen in Table 3, considerable animalto-animal variability was detected. Whereas all mice used VH 7183 and 5558 to encode anti-DNA antibodies, the frequency with which these families were utilized in different animals varied from 11% to 36% for VH 7183 and from 20% to 65% for VH 5558. In addition, 4 of the 6 animals examined utilized 5606, 2 each utilized S107 and 36-60, and only 1 utilized X24 or 3609 to produce IgG anti-DNA antibodies. Individual mice used 2-5 different VH families to encode antiDNA antibodies. This represents a minimum estimate of the diversity of VH family usage, since not all families were examined. DISCUSSION This study examined the frequency with which specific VH families contribute to the production of VH FAMILY USAGE BY ANTI-DNA-PRODUCING CELLS IgG anti-DNA antibodies in autoimmune mice. Initial studies confirmed that the size, number, and complexity of the VH repertoire expressed by MRLllpr mice was similar to that reported for normal animals (Table 2 and refs. 39, 41, and 42). The analysis of 468 anti-DNA-secreting colonies revealed that each of the 7 VH families examined in this investigation could encode IgG anti-DNA. As a general rule, individual families were utilized at a frequency roughly proportional to their representation in the MRLlIpr repertoire as a whole (Table 2). There were 2 exceptions to this generalization: The 7183 family was overutilized (P< O.OOl), while the 36-60 family was underutilized (P < O.OOl), for anti-DNA production. Although all MRLlIpr mice used the 5558 and 7183 families to produce IgG anti-DNA, the frequency with which these families were used varied considerably among individual animals. VH 5558 contributed 20-65%, and VH 7183 contributed 11-36%, of the total DNA-specific response of different mice (Table 3). Other VH families were used with even less consistency: Only 4 of 6 animals used J606,2 of 6 used S107 or 36-60, and only 1 of 6 used X24 or 3609 to encode IgG anti-DNA. The high concentration of serum autoantibodies in lupus mice has led some investigators to postulate that Ig gene abnormalities play a role in disease pathogenesis (39,43). Classic mendelian analysis of Ig heavy chain allotypes and Southern blot analyses using VH-specific probes have revealed no linkage between SLE and polymorphisms at the H chain complex (4446). However a number of studies have shown that D-proximal VH gene families (7183 and Q52) were utilized preferentially by neonatal B cells (41,47-51), and there is some evidence that these families are overutilized by autoimmune mice (24,52). The latter finding is controversial, since Kastner et a1 found no evidence that 3’ VH families were systematically overexpressed in mice with autoimmune disease (39). It was concluded that VH gene utilization by MRWlpr mice was stochastic-a finding consistent with that of Teale and Morris, who reported that VK light chain gene usage was random in this strain (42). There is considerable controversy concerning the mechanism(s) responsible for the initiation and perpetuation of autoantibody production in SLE. Functional studies of B cell isotype, idiotype, and cross-reactivity indicate that many different clones may be stimulated to secrete anti-DNA antibodies in a single animal (13,34,53,54), but this result has been challenged by molecular evidence suggesting that 565 antigen-reactive B cells from individual mice may be clonally related (21,27,55). More recently, it was shown that disease in Ipr mice results from defective expression of the f u s gene product (56)-an abnormality that presumably affects B cells of all specificities. These observations have raised questions concerning the process(es) responsible for selecting and diversifying the IgG anti-DNA repertoire. To analyze the diversity of the VH families contributing to the production of IgG anti-DNA antibodies, previous investigators generated panels of hybridomas. Recent evidence suggests that lymphocytes secreting IgG autoantibodies in vivo do not readily form stable hybrids (30), which may explain why relatively few IgG anti-DNA-secreting hybridomas have been cloned from any single mouse (22,2427,52). Indeed, those hybridomas that have been isolated may represent an atypical or pauci-clonal subset of a more diverse population of in vivo-activated B cells (29). In this context, it has been shown that IgG autoantibody-secreting hybridomas do not accurately reflect the autoantibody specificities present in the serum of the mice from which they were derived (30). To avoid these difficulties and generate a larger and potentially more representative repertoire of autoantibody-secreting B cells, we adopted the method of Kelsoe (31,32). B cell colonies were produced by short-term culture of MRLllpr B cells in the presence of LPS. Using this approach, an average of 61 IgG anti-DNA-secreting clones were derived per 5 X lo6 splenic lymphocytes per animal. This cloning efficiency is 1-2 orders of magnitude higher than that reported for the production of IgG anti-DNAsecreting hybridomas following cell fusion (20-26). The culture system reported by Kelsoe yielded Ig-secreting clones whose antigenic specificity was similar to that of in vivo-activated B cells (Table 1 and data not shown). Moreover, previous reports showed that cells induced to secrete IgG following LPS stimulation were derived from in vivo-activated precursors (33). Other investigators have shown that VH family usage by B cells cultured with LPS is similar to that of normal unstimulated lymphocytes (42). Thus, while some degree of repertoire selection might take place during short-term culture (a disproportionate fraction of DNA-specific clones produced IgM in vitro), evidence suggests that the colonies secreting IgG anti-DNA antibodies were derived from B cells that had been actively producing Ig of that specificity and isotype in vivo (33). We found that the VH repertoire expressed by KLINMAN ET AL 566 MRLllpr mice was of normal size and complexity, and that all VH families could contribute to the production of IgG anti-DNA antibodies. Our analysis of 40-170 IgG anti-DNA-secreting colonies per mouse revealed that 2-5 different VH families encoded IgG anti-DNA antibodies in each animal (mean -+ SD 3.7 -+ 1.0) and that no animal derived more than 2745% of its antiDNA response from a single VH family (Table 3). Since up to 30% of the anti-DNA-secreting colonies examined did not hybridize with any of the 7 VH probes used, this represents a minimum estimate of the number of VH families involved in the anti-DNA response. These data can be compared with those derived from studies of hybridomas. Shlomchik et a1 analyzed 4-6 IgG anti-DNA-secreting hybridomas from each of 5 MRLllpr mice (21,27). They found that 1) only 1-2 (mean ? SD 1.6 +- 0.5) VH families were involved in the production of IgG anti-DNA antibodies in a single animal, and 2) hybridomas from individual mice using the same VH family were always clonally related. By comparison, Tillman et al analyzed a mean (+SD) of 7.6 ? 6.7 IgG anti-DNA-secreting hybridomas from 9 NZB X NZW mice (25). They found that 1-4 (mean 2.1 1.1) VH families were involved in the production of these antibodies, and that multiple members of a single family were used to produce anti-DNA antibodies (25). The latter finding has been confirmed by other investigators (26). Our analysis of a much larger number of antiDNA-secreting colonies per animal supports the conclusion of Tillman et a1 that the anti-DNA response is oligoclonal. In contrast, our data differ from those of Shlomchik et al, and do not support the conclusion of those investigators that this response is pauci-clonal or monoclonal. Clonal diversity among IgG anti-DNAsecreting cells is consistent with the functional heterogeneity of anti-DNA antibodies (13,34,53,54) and the complexity of the DNA molecule itself, which, unlike a simple hapten, is composed of multiple immunogenic epitopes (57). Other investigators have also evaluated VH family utilization in mice with lupus. PanosianSahakian et a1 (20) generated 18 anti-DNA hybridomas from a single NZB x NZW mouse and found that they were derived from a minimum of 12 different VH genes from 6 different VH families. Bona studied a pool of 23 DNA-reactive hybridomas from a group of unrelated mice and found that they were encoded by the J558, 7183, Q52, and S107 families (52). Smith and Voss (22) reported that 5 monoclonal anti-DNA antibodies from * NZB x NZW mice utilized 3 different VH families, and Trepicchio and Barrett determined that 11 monoclonal anti-DNA antibodies from MRLllpr mice were derived from at least 9 different VH genes selected from 4 VH families (24). These findings are consistent with our observation that the IgG anti-DNA response in individual mice can involve multiple different clones and VH families. Unfortunately, we were unable to generate sequence data from the small number of cells present in LPS-stimulated anti-DNA-secreting colonies. However, other investigators have shown that multiple members of the same VH family are used to encode IgG anti-DNA antibodies in a single mouse (25,26). 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