Severe impairment of B cell function in lpr/lpr mice expressing transgenic Fas selectively on B cells

Hajime Komano1, Yuko Ikegami1, Minesuke Yokoyama2, Rika Suzuki2, Shin Yonehara3, Yoshiki Yamasaki4 and Nobukata Shinohara1,5

1 Department of Immunology and
2 Reproductive Engineering Section, Mitsubishi Kasei Institute of Life Sciences, Machida, Japan
3 Institute for Virus Research, Kyoto University, Kyoto, Japan
4 The Pharmaceutical Basic Research Laboratories, JT Inc., Yokohama, Japan
5 Department of Immunology, Kitasato University School of Medicine, Sagamihara, Kanagawa 228, Japan

Correspondence to: N. Shinohara, Department of Immunology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transgenic lpr/lpr mice expressing functional Fas selectively on B cells were produced in an attempt to elucidate the role of Fas on B cells in the regulation of autoantibody production. The homozygous lpr/lpr mice carrying the transgene did not produce anti-double-stranded DNA antibodies throughout their lives, whereas the development of abnormal lpr T cells (double negative, B220+) was not suppressed. Further analyses, however, revealed that the expression of the transgenic Fas on B cells of lpr/lpr homozygous mice resulted in severe impairment of the B cell function. The defect was characterized by a decrease in the number of mature peripheral B cells, a reduction in the serum Ig level and the total failure of B cells to mount antibody responses to stimulations of T-dependent as well as T-independent antigens. Such a defect was prominent only when the transgene was expressed on the lpr/lpr homozygous background. On the contrary, B cells of the transgenic lpr/lpr mice were shown to be capable of producing Ig when stimulated with anti-CD40 in the presence of IL-4 and IL-5. Furthermore, lpr/lpr T cells showed enhanced non-specific cytolytic activity. These observations suggested that the observed B cell defect was probably attributable to the destruction of activated B cells expressing transgenic Fas by aggressive lpr/lpr T cells.

Keywords: autoantibody, B cell, Fas, lpr


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice homozygous for a defective Fas structural gene (lpr) develop a disease characterized by autoantibody production and massive proliferation of abnormal double-negative T cells (1). A very similar disease develops in mice carrying a defective Fas ligand (FasL) gene (gld) (2). These mutant mice indicate a crucial role of the FasL–Fas interactions in the regulation of autoantibody production. Nevertheless, the precise etiological mechanism underlying autoantibody production in these mice is unclear. A number of studies have been focused on possible failure in elimination of autoreactive T cells due to the lack of functional Fas on the cells of the T lineage (38), without a clear conclusion. Several lines of studies employing cell transfer experiments indicated that the intrinsic defect of B cells is the primary cause of induction of autoantibody production in lpr mice (911). A recently proposed theory concerning the regulation of autoantibody production mediated by the cytolytic CD4+ T cells predicts that expression of functional Fas on B cells rather than T cells is essential for the regulation of autoantibody production (12). Indeed, transgenic expression of Fas on T cells of lpr/lpr mice did not shut off autoantibody production, whereas the development of the abnormal T cells was completely inhibited (9), indicating that the abnormality of lpr T cells is not the direct cause of autoantibody production. Although another group reported that T-restricted expression of Fas in lpr mice inhibited both abnormalities, the presented data indicated that autoantibody production remained definitely positive albeit reduced (13). On the other hand, it has been shown that T cells of lpr mice express high levels of FasL mRNA (14), suggesting aggressiveness of such T cells. Such enhanced cytolytic activity of lpr T cells might have complicated the results of certain cell transfer experiments. Considering these problems, we developed transgenic lpr/lpr mice expressing Fas selectively on B cells in an attempt to elucidate the role of Fas on B cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transgenic construct
A 9.6 kb fragment of a rearranged genomic Ig {kappa} light chain gene of anti-TNP IgM (15) was subcloned into pBluescript SK(–) and an EcoRI site was introduced at the first codon by site-directed mutagenesis. BALB/c-derived full-length Fas cDNA was subcloned at the introduced EcoRI site in the orthodromic orientation. The purified 11.1 kb BamHI fragment was microinjected into fertilized mouse eggs obtained between C3H/HeJ females and C3H/HeJ-lpr males.

Typing of mice
PCR was carried out on DNA extracted from tails. Following an initial denaturation step (94°C, 4 min), amplification was carried out through 30 cycles at 94°C for 45 s, 55°C for 1 min and 72°C for 1 min. The primers used for detecting the transgene were: 5' primer, 5'-CCATCGATCGCCGCCATGGGAAAGTACCGGAAAAGAAAG-3'; and 3' primer, 5'-CCATCGATGGCTCCAGACATTGTCCTTCATTTTCATTT-3'. The wild-type and the lpr alleles of the Fas gene was examined by PCR using a sense primer, 5'-AGGTTACAAAAGGTCACCC-3', of intron 2 and two antisense primers, 5'-GATACGAAGATCCTTTCCTGTG-3' and 5'-CAAACGCAGTCAAATCTG- CTC-3', of the early transposable element (Etn) and intron 2 respectively (1). PCR conditions were the same as those described above. PCR products were gel electrophoresed, stained with ethidium bromide and photographed with UV illumination. The 500 bp fragment indicates the transgene. The 560 and 180 bp fragments correspond to lpr mutant and wild-type Fas genes respectively.

Flow cytometry
RMF2, a monoclonal anti-Fas antibody specific for the BALB/c allele (16), was used to detect the transgenic Fas, and Jo2, non-allelic anti-Fas mAb (16), was used to determine total amounts of expressed Fas including endogenous and transgenic products. FITC–anti-CD4, FITC–anti-CD8, FITC–anti-Thy1, phycoerythrin (PE)–anti-Thy-1, PE–anti-B220, biotin–anti-IgD and FITC–anti-IgM were purchased from PharMingen (San Diego, CA). PE–streptavidin was purchased from Sigma (St. Louis, MO). Cells (2–10x105) were stained in and washed with ice-cold Hank's balanced salt solution containing 0.5% BSA and 0.02% sodium azide. Stained cells after washing were examined by flow cytometric analyses on FACScan (Becton Dickinson, Mountain View, CA).

Titration of anti-DNA antibody
Microtiter plates were coated with 10 µg/ml antigen solution overnight at 4°C. Purified mouse genomic DNA was used as antigen. Wells were washed with PBS containing 0.05% Tween 20, blocked with diluted normal rabbit serum for 1 h at 25°C and then incubated with 50 µl of serially diluted serum samples for 2 h at 25°C. Washed wells were further incubated with horseradish peroxidase-labeled rabbit anti-mouse Ig antiserum for 1h at 25°C. Colorimetric analyses were carried out using tetramethylbenzidine (TMB) as the substrate.

Immunization and titration of antibody
Mice were immunized by i.p. injection of 5x108 sheep red blood cells (SRBC), two biweekly injections of 100 µg of TNP-keyhole limpet hemacyanin (KLH) in complete Freund's adjuvant, or 100 µg of TNP-Ficoll and bled on day 3, 7 or 5 respectively of the last immunization. The titers of antibodies to SRBC were determined by hemagglutination. Anti-TNP antibodies were measured by ELISA on TNP-BSA-coated plates. Polyvalent anti-mouse Ig antibody labeled with horseradish peroxidase (HRP) was used as a secondary reagent. TMB was used as the substrate.

In vitro production of Ig by B cells
Nylon wool-adherent spleen cells were treated with anti-Thy-1 antibody and complement. After the treatment, viable cells were recovered by centrifugation on Ficoll. The cells were stimulated by 10 µg/ml of anti-CD40 antibody in flat-bottomed wells of 96-well plates in the presence of 100 U/ml of IL-4 and IL-5. Amounts of Ig in day 3 culture supernatants were determined by ELISA using affinity-purified polyvalent rabbit anti-mouse Ig antibodies as coating and developing (HRP-labeled) reagents.

Cell-mediated cytotoxicity assay
The nylon wool-non-adherent splenic T cell fraction was used as an effector population. CML assays were performed as described previously (18). Briefly, graded numbers of effector cells were incubated with 5x103 51Cr-labeled A20.2J cells (Fas + BALB/c B cell tumor) for 6 h at 37°C in 10% CO2. Percent specific lysis was calculated as (experimental release – spontaneous release)/(maximal release – spontaneous release)x100.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Development of mice expressing transgenic Fas selectively on B cells
Full-length BALB/c-derived Fas cDNA was inserted at the head of the leader exon of a rearranged genomic murine Ig {kappa} light chain gene (Fig. 1aGo), expecting that defined and undefined regulatory elements present within the {kappa} structural gene would provide regulations necessary for B cell-specific expression. The construct was injected into single-cell embryos fertilized in vitro between C3H/HeJ-lpr males and C3H/HeJ females. A transgenic male expressing the transgenic Fas molecules on lymphocytes was obtained, back-crossed to C3H and C3H-lpr/lpr females, and resulting offspring were analyzed for Fas expression.



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Fig. 1. The construct for generating B cell-specific Fas transgenic mouse (A) and genotyping of mice by PCR (B). BALB/c-derived full-length Fas cDNA was inserted into a 9.6 kb fragment of a rearranged genomic Ig {kappa} light chain gene of anti-TNP IgM at the first codon. PCR was carried out on DNA extracted from tails. The 500 bp fragment indicates the transgene. The 560 and 180 bp fragments indicate lpr mutant and wild-type Fas genes respectively. See Methods for the primers and conditions used in PCR.

 
An allele-specific anti-Fas (BALB/c) mAb, RMF2 (16), and a pan anti-Fas mAb, Jo2 (16), were used to detect transgenic and total Fas molecules respectively. In spleens of transgenic C3H mice carrying no lpr defect, the transgenic Fas was detected on sIgM+ cells but not on sIgM cells (Fig. 2aGo). Accordingly, the transgenic Fas was not detectable on Thy-1+ cells (data not shown). It is worth mentioning that both {kappa} and {lambda} type B cells equally expressed the transgenic Fas indicating that the transgenic construct did not confer light chain isotype-specific expression. Furthermore, the transgenic Fas was not detectable on the thymocytes either, whereas endogenous Fas was normally expressed (Fig. 2bGo). These results indicate that the transgenic Fas was indeed expressed on B cells but not on cells of the T lineage. Unfortunately, this transgene turned out to be lethal and thus the transgenic mice were maintained as heterozygotes with individual typing.



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Fig. 2. B cell-specific expression of transgenic Fas molecules in non-lpr mice. Peripheral blood lymphocytes (a) and thymocytes (b) from transgenic and non-transgenic C3H mice were analyzed for expression of transgenic Fas molecules by flow cytometry. RMF2, a monoclonal anti-Fas antibody specific for the BALB/c allele, was used to detect the transgenic Fas and Jo2, non-allelic anti-Fas mAb, was used to examine the total amount of expressed Fas including endogenous and transgenic products. Peripheral blood lymphocytes were stained with FITC-labeled anti-IgM mAb and biotinylated RMF2 developed by PE–streptavidin. Thymocytes were stained with Jo2 or RMF2. NS stands for negative staining where only the secondary fluorescent reagent was added. In total, 5x103 events, gated by forward and light scatter.

 
Development of lpr T cells and reduction of mature B cells in transgenic lpr/lpr mice
Mice carrying the transgene were backcrossed to C3H-lpr mice and transgene-positive lpr/lpr mice were obtained. The transgenic lpr/lpr mice developed lymphadenopathy comparable to that of non-transgenic lpr/lpr mice in 4 months. The enlargement of lymphoid organs was due to massive proliferation of B220+ T cells (Fig. 3Go, left column, boxed population). These T cells were CD4/CD8 double negative and indistinguishable from those found in non-transgenic lpr/lpr mice. Such abnormal T cells were already detected at the age of 8 weeks. This result is consistent with the lack of the transgene expression on the cells of T lineage.



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Fig. 3. Staining profiles of lymphocytes of transgenic lpr/lpr mice. Spleen cells from C3H [TG(–/–) lpr(w/w)], transgenic C3H [TG(+/–) lpr(w/w)], C3H-lpr [TG(–/–) lpr(l/l)] and transgenic C3H-lpr [TG(+/–) lpr(l/l)] mice were stained with FITC–anti-Thy-1 and biotinylated anti-B-220 + PE–avidin (left panels) or with FITC–anti-IgM and biotin-anti-Fas (Jo2) + PE–avidin (right panels). The presence of abnormal B220+, Thy-1+ T cells (boxed population) was revealed in spleens of transgenic C3H lpr/lpr mice (left panels). In the spleens of transgenic lpr/lpr mice, numbers of B cells were significantly reduced. Nevertheless, the enhanced expression of Fas was evident on the residual B cells (right panel: second top versus bottom, boxed population). In total, 1x104 events were analyzed by FACScan (Becton Dickinson) with gating for lymphocytes by forward and side scatter. The contour lanes represent 5, 10, 20, 40, 80 and 160 cells, respectively.

 
The transgenic lpr/lpr mice showed a significant reduction in the number of sIgM+ B cells in peripheral lymphoid organs compared with non-transgenic lpr/lpr littermates (Fig. 3Go, right column, boxed population). Absolute numbers of sIgM+ cells recovered from mesenteric lymph nodes and spleens of transgenic lpr/lpr mice (0.1x106 and 13x106 on average respectively) were significantly lower than those of non-transgenic lpr/lpr littermates (0.88x106 and 54.7x106 respectively). Thus the number of B cells in the transgenic lpr/lpr was ~10–20% of the non-transgenic counterparts. Although the residual B cells in the transgenic lpr/lpr mice expressed significant levels of transgenic Fas, depletion was more prominent with high expressers (Fig. 3Go, right column, fourth panel, boxed population). Such a decrease in the B cell number was not observed in transgenic mice without the lpr defect, i.e. non-lpr and lpr heterozygous mice.

In order to study cells of the B lineage in early developmental stages, bone marrow cells of the transgenic lpr/lpr mice were analyzed. As shown in Fig. 4Go, the numbers of pre-B cells (sIgM, B220+) and newly generated B cells (sIgM+, B220+) in transgenic lpr/lpr bone marrow were comparable to those in non-transgenic lpr/lpr bone marrow. Thus, the development of B cells in the bone marrow of transgenic lpr/lpr mice was not affected by the expression of the transgenic Fas. In contrast, a significant decrease in the number of mature B cells (sIgM+, B220high) was observed in transgenic lpr/ lpr bone marrow. Also, very few sIgM+, IgDhigh B cells were detected in bone marrow of transgenic lpr/lpr mice (data not shown). Since B220high, IgDhigh B cells found in bone marrow represent recirculating B cells (19,20), the observed depletion of B cells must have occurred after maturation, possibly in peripheral organs. Pre-B cells and newly generated B cells of the transgenic mice also expressed Fas on their surface in amounts comparable to that on transgenic peripheral B cells (data not shown).



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Fig. 4. B cell development in the bone marrow of a transgenic lpr/lpr mouse. Population of Pre-B cells (IgMB220lo) and immature B cells (IgM+B220lo) in the bone marrow showed no difference between transgenic and non-transgenic lpr/lpr mice, whereas the number of mature B cells (IgM+B220hi) was significantly reduced in the transgenic mice (top panels). Single-cell suspensions were prepared and stained with antibodies. The following antibodies were used: FITC–anti-CD4, FITC–anti-CD8, FITC– and PE–anti-Thy1, biotinylated and PE–anti-B220, biotinylated anti-IgD, and FITC–anti-IgM. Biotinylated antibodies were detected by streptavidin–PE. In total, 1x104 events were analyzed by FACScan (Becton Dickinson) with gating for lymphocytes by forward and side scatter.

 
Ig level and anti-double-stranded (ds) DNA antibodies in transgenic lpr/lpr mice
Quantitation of serum Ig levels showed 5- to 10-fold decrease in total Ig level (Fig. 5Go) equally involving all isotypes and both {kappa} and {lambda} types (data not shown) in transgenic lpr/lpr mice. The resulting serum Ig level of the transgenic lpr/lpr was comparable to that of the normal C3H/HeJ mouse.



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Fig. 5. Serum levels of Ig and the absence of anti-dsDNA reactivity in sera of transgenic lpr/lpr mice. Mice were bled at 7 months of age. (Left) Total serum Ig levels were determined by {kappa} light chain isotype-specific ELISA (PharMingen). Serum levels of Ig of individual normal C3H (open circle), non-transgenic (closed circle) and transgenic lpr/lpr mice (closed square) are shown. Serum levels of individual isotypes showed that Ig of all isotypes were similarly affected by the introduction of the transgene (data not shown). (Right) Serum anti-dsDNA antibodies measured by ELISA. Symbols are the same as those in the left panel. No anti-dsDNA reactivity was detected in transgenic sera. Almost identical results were obtained in another set of mice (8 months old) which included five phenotypically normal (TG–/– w/lpr), six TG+/– w/w, four TG–/– lpr/lpr and three TG+/– lpr/lpr mice. In this set too, all five phenotypically normal mice produced low but significant levels of anti-DNA antibody, whereas none of the three TG+/– lpr/lpr mice produced detectable amounts of the antibody.

 
Serum levels of anti-dsDNA antibodies of transgenic and non-transgenic lpr/lpr littermates were determined at 3, 5 and 7 months of age. Two sets of the experiments were performed at the age of 7 and 8 months of age with almost identical results, and Fig. 5Go shows the result of one experiment at 7 months. Anti-dsDNA titers of individual mice grouped according to their genotypes are expressed. The serum levels of anti-dsDNA antibody remained undetectable, i.e. even lower than the level of normal mouse serum, in the transgenic lpr/lpr mice throughout their lives. This difference was reproduced in another set of the experiments (see legend to Fig. 5Go). Very low but significant amounts of anti-dsDNA antibodies were reproducibly detected in normal mouse sera. Since the anti-DNA antibody titers of transgenic lpr 800 /lpr were, if any, <1% of those of non-transgenic counterparts while their total Ig levels were ~10%, the virtual absence of the autoantibody in transgenic mice cannot be explained solely by the general decrease in Ig production.

Failure of transgenic lpr/lpr mice to mount antibody responses
The responsiveness of transgenic B cells to a variety of antigenic stimulations was studied. Littermates obtained by crossing between non-transgenic lpr heterozygous (w/l) females and Fas-transgene (TG) heterozygous (+/–) lpr homozygous (w/l) males were immunized with sheep red blood cells (SRBC), and serum anti-SRBC IgM antibody titers were determined by hemagglutination on day 3. Figure 6Go shows the results summarized into four groups according to the genotypes of the mice with respect to the transgenic Fas gene (TG) and the lpr locus (endogenous Fas gene). The day 3 anti-SRBC IgM antibody titers of non-transgenic (–/–) lpr heterozygous (w/l) and lpr homozygous (l/l) mice were comparable (left panel), which were also comparable to those of wild-type C3H mice (data not shown). Although TG(+/–) lpr(w/l) mice also produced comparable amounts of the antibody, none of the TG(+/–) lpr(l/l) mice produced detectable amounts of the antibody. Anti-SRBC antibodies remained undetectable in these mice for 2 weeks and even after a secondary immunization (data not shown).



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Fig. 6. Failure of transgenic lpr/lpr mice to mount antibody responses. (Left) Littermates obtained by crossing between non-transgenic lpr homozygous (l/l) females and lpr heterozygous (w/l) Fas-transgene (TG) heterozygous (+/–) males were immunized by an i.p. injection of 1x108 SRBC and serum anti-SRBC IgM antibody (2-mercaptoethanol-sensitive) titers were determined by hemagglutination on day 3. (Center) Other littermates were immunized with TNP-KLH in complete Freund's adjuvant twice with a 14 day interval and anti-TNP IgG antibody titers were determined 7 days after the second immunization. (Right) Offspring of [TG(+/–) lpr(l/w)xTG(+/–) lpr(l/w)] crosses were immunized with TNP-Ficoll and anti-TNP antibody titers of day 5 sera were determined. Genotypes of individual mice are shown below the graph.

 
A similar experiment was performed on another set of mice using TNP-KLH as an antigen in complete Freund's adjuvant. In this experiment, animals were immunized twice with a 14 day interval and anti-TNP IgG antibody titers were determined 7 days after the second immunization. As also shown in Fig. 6Go, TG(+/–) lpr(l/l) mice again failed to mount significant responses in the secondary IgG antibody production while mice of other genotypes produced antibodies. Although the antibody responses of TG(+/–) lpr(w/l) mice were slightly lower than those of transgene-negative responders, none of them showed such marked unresponsiveness as seen in TG(+/–) lpr(l/l) mice. The amounts of antibodies produced by TG(–/–) lpr(l/w) mice were not different from those produced by normal C3H mice.

Finally, the antibody response of the transgenic mouse to a T-independent antigen which does not involve cognate T–B interactions was studied. Offspring of [TG(+/–) lpr(l/w)x TG(+/–) lpr(l/w)] crosses were immunized with TNP-Ficoll and anti-TNP antibody titers of day 5 sera were determined (Fig. 6Go). In this T-independent antibody response too, TG(+/–) lpr(l/l) mice were incapable of mounting any significant responses. TG(+/–) lpr(w/w) mice mounted lower but significant responses.

These experiments revealed a severe impairment of B cell responses in TG(+/–) 900 lpr(l/l) mice. Therefore, the complete absence of anti-dsDNA antibody observed in TG(+/–) lpr(l/l) mice probably reflects this general impairment of B cell responsiveness rather than a specific defect in the surveillance of autoantibody production. Interestingly, the suppressive effect of the transgene expression was prominent only on the lpr homozygous background and was not obvious or was much less significant in TG(+/–) lpr(w/l) or TG(+/–) lpr(w/w) mice.

Normal non-specific Ig production by transgenic lpr/lpr B cells
In spite of the almost complete unresponsiveness of the TG(+/–) lpr(l/l) B cells to antigenic stimulations, the serum Ig levels of these mice appeared to match the number of residual mature B cells. Therefore ability of these B cells to produce Ig to non-antigenic stimulations was studied. Nylon wool-adherent splenic lymphocytes prepared from normal C3H and TG(+/–) lpr(l/l) spleens were further depleted for T cells by anti-Thy-1 antibody and complement treatment. Graded numbers of the cells were cultured in the presence of anti-CD40 antibody, IL-4 and IL-5. Figure 7Go shows amounts of Ig detected in day 3 culture supernatants. On the left graph of Fig. 7Go, the amount of Ig was plotted against the number of viable nucleated cells seeded in cultures. The right graph shows adjusted plotting which indicates the amounts of Ig plotted against the number of B cells present in the cultures as calculated from flow cytometric determination. These graphs indicate that TG(+/–) lpr(l/l) B cells were capable of producing amounts of Ig comparable to those by normal B cells.



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Fig. 7. Ig production of TG(+/–) lpr(l/l) lymphocytes. Nylon wool-adherent spleen cells of normal C3H and TG(+/–) lpr(l/l) mice were cultured in the presence of anti-CD40 mAb, IL-4 and IL-5. The amount of Ig in the culture supernatant was determined by ELISA using polyvalent anti-IgG (anti-{gamma} chain + anti-light chain) antibody. (Left) Amount of Ig/viable cells cultured. (Right) The data shown in the left panel are expressed as amount of Ig/B cells in culture. The B cell content of each cell preparation was determined by flow cytometry and the percent value obtained was used for the adjustment.

 
Aggressive lpr T cells
Since lpr T cells are known to express high levels of FasL mRNA (14), it appeared likely that B cells expressing transgenic Fas became targets for aggressive lpr T cells after the antigenic stimulation. Therefore, lytic activity of lpr/lpr T cells was studied. Nylon wool-non-adherent splenic lymphocytes further depleted for class II MHC positive cells were used as T cells (>85% of nucleated cells were Thy-1+ cells). Both TG(+/–) lpr(l/l) splenic T cells and normal splenic T cells showed lytic activities on Fas+ B cell tumor, A20.2J. However, the lytic activity of TG(+/–) lpr(l/l) was higher than that of normal T cells by 4-fold. Addition of anti-CD3 antibody enhanced the lytic activities of both cell populations. In this situation, TG(+/–) 1000 lpr(l/l) also showed higher lytic activity. The lytic activity of TG(+/–) lpr(l/l) T cells was sensitive to complement-dependent cytotoxic treatment by anti-B220 antibody but not to anti-CD8 nor to anti-CD4. Thus the vast majority of the lytic activity was attributable to double-negative B220+ lpr T cells. The 4-fold higher lytic activity of TG(+/–) lpr(l/l) T cells compared with that of normal T cells and the presence of a huge number of abnormal lpr T cells in lpr/lpr mice probably explain the different behaviors of Fas-transgenic B cells between lpr homozygote and non-lpr mice including heterozygotes.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The transgenic lpr/lpr mice expressing Fas selectively on B cells were produced in an attempt to elucidate the role of Fas on B cells in the regulation of autoantibody production. The homozygous lpr/lpr mice carrying the transgene failed to produce anti-dsDNA antibodies throughout their lives, whereas the development of abnormal lpr T cells was not inhibited. At a glance, the result apparently suggested a crucial role played by Fas on B cells in the surveillance of autoantibody production (9). However, more detailed analyses revealed that the absence of autoantibody production most likely reflected the general impotency of the transgenic B cells in mounting specific antibody responses in the lpr/lpr environment. Therefore, these transgenic mice turned out to be inappropriate for our original purpose.

The introduction of the transgene into lpr/lpr background resulted in a defective B cell population. The defect of B cells in transgenic lpr/lpr mice was characterized by a decrease in the number of peripheral mature B cells and the failure to mount specific antibody responses. We speculate that these defects are the consequence of the attack of the transgenic B cells by aggressive lpr T cells. The speculation is based on the following observations. (i) These signs were obvious only in the lpr/lpr environment and merely a marginal or moderate reduction of antibody responses was observed in non-lpr/lpr transgenic mice. (ii) Ig production by the TG(+/–) lpr(l/l) B cells in response to a non-specific stimulation by anti-CD40 antibody was comparable to that by normal B cells. (iii) lpr/lpr T cells were shown to have high non-specific cytolytic activity. Thus, it is likely that activated B cells expressing the transgenic Fas suffered non-specific attack of the aggressive T cells in the 1100 lpr/lpr environment. Although this situation was generated by transgenic expression of Fas, the observation suggests that Fas-expressing activated B cells can be attacked by lytic T cells in vivo. Indeed, Rathmell et al. showed that anergic autoreactive B cell clones were eliminated in vivo by Fas-based attack by T cells (21). The suppressive effect in these mice was observed on the T independent antibody response as well as on T-dependent responses, implying that cognate T–B interactions were not essential for this phenomenon. The suppression was almost complete in TG(+/–) lpr(l/l) mice, whereas the Ig production of the residual B cells was normal and serum Ig level matched the number of peripheral B cells. These observations suggest that the sensitivity of transgenic B cells to Fas-mediated lysis increased after antigenic stimulations, i.e. sIg-mediated activation. In this respect, it is interesting that spontaneous anti-DNA antibody production was completely abrogated in TG(+/–) lpr(l/l) mice, suggesting that the apparently spontaneous production of this antibody probably depends on antigenic stimulations. In addition, the presence of low but significant amounts of anti-dsDNA antibody in the sera of normal mice indicates that production of a small amount of such antibodies is not abnormal. The abnormality of lpr mice is the uninhibited production of a large amount of autoantibodies.

Two different theories have been proposed to explain the mechanism of autoantibody production in lpr/lpr mice. One proposes a failure in eliminating or shutting down self-reactive T cells as the essential cause and the other blames B cells for having an intrinsic defect. Although functional abnormality of mature T cells in lpr/lpr mice has been indicated, the negative selection of autoreactive T cell clones has been shown to take place in the thymus of lpr/lpr mice (38). A recent report showed that transgenic expression of Fas on T cells alone abrogated the massive proliferation of abnormal T cells, whereas it did not stop the production of anti-dsDNA antibody (9). Thus, the observations indicate that autoantibody production and development of lpr T cells are two separate abnormalities caused by the Fas defect. Cell transfer analyses showed that only lpr B cells produced autoantibodies when co-transferred with normal B cells together with lpr T cells, indicating a key contribution of an intrinsic defect of B cells rather than T cells to autoantibody production in lpr/lpr mice (11,12). Considering the observations in this study, however, the results might be attributed to the aggressiveness of lpr T cells and the sensitivity of normal B cells to the attack by such T cells. Thus the solution to the two conflicting opinions is yet to be available. We are now in the process of developing mice expressing dominant negative mutant Fas on selected cell populations, hoping to obtain a clearer answer to this problem.



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Fig. 8. Enhanced lytic activity of lpr/lpr T cells. (Top) Nylon wool-non-adherent spleen cells of normal C3H and lpr/lpr mice were tested on A20.2J, a Fas-expressing B lymphoma of BALB/c origin. The assays were carried out in the presence or absence of 1 µg/ml of anti-CD3 antibody (2C11). The 6 h 51Cr release was determined. (Bottom) T cells of the lpr/lpr mouse were treated with indicated antibody and complement before the assay.

 

    Acknowledgments
 
We thank A. Nakamura for secretarial work, and also thank S. Kamijo and his colleagues for taking care of animals. All experiments were performed in accordance with institutional guidelines.


    Abbreviations
 
dsdouble stranded
FasLFas ligand
HRPhorseradish peroxidase
KLHkeyhole limpet hemacyanin
PEphycoerythrin
SBRCsheep red blood cell
TMBtetramethylbenzidine

    Notes
 
Transmitting editor: K. Okumura

Received 24 November 1998, accepted 10 March 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Watanabe-Fukunaga R., Brannan, C. I., Copeland, N. G., Jenkins, N. A. and Nagata, S. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314.[ISI][Medline]
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