Cells and Dendritic Cells Localization of Anti-Double-Stranded DNA

Fas/Fas Ligand Deficiency Results in Altered Localization of Anti-Double-Stranded DNA B Cells and Dendritic Cells

This information is current as Michele L. Fields, Caroline L. Sokol, Ashlyn Eaton-Bassiri, of October 2, 2021. Su-jean Seo, Michael P. Madaio and Jan Erikson J Immunol 2001; 167:2370-2378; ; doi: 10.4049/jimmunol.167.4.2370 http://www.jimmunol.org/content/167/4/2370 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Fas/Fas Ligand Deﬁciency Results in Altered Localization of Anti-Double-Stranded DNA B Cells and Dendritic Cells1

Michele L. Fields,* Caroline L. Sokol,* Ashlyn Eaton-Bassiri,* Su-jean Seo,* Michael P. Madaio,† and Jan Erikson2*

Autoantibodies directed against dsDNA are found in patients with systemic lupus erythematosus as well as in mice functionally deficient in either Fas or Fas ligand (FasL) (lpr/lpr or gld/gld mice). Previously, an IgH chain transgene has been used to track anti-dsDNA B cells in both nonautoimmune BALB/c mice, in which autoreactive B cells are held in check, and MRL-lpr/lpr mice, in which autoantibodies are produced. In this study, we have isolated the Fas/FasL mutations away from the autoimmune-prone MRL background, and we show that anti-dsDNA B cells in Fas/FasL-deficient BALB/c mice are no longer follicularly excluded, and they produce autoantibodies. Strikingly, this is accompanied by alterations in the frequency and localization of dendritic cells as well as a global increase in CD4 T cell activation. Notably, as opposed to MRL-lpr/lpr mice, BALB-lpr/lpr mice show no Downloaded from appreciable kidney pathology. Thus, while some aspects of autoimmune pathology (e.g., nephritis) rely on the interaction of the MRL background with the lpr mutation, mutations in Fas/FasL alone are sufficient to alter the fate of anti-dsDNA B cells, dendritic cells, and T cells. The Journal of Immunology, 2001, 167: 2370Ð2378.

he presence of serum autoantibodies directed against nu- end, the lpr and gld defects were bred onto the VH3H9 BALB/c clear Ags such as dsDNA is one of the hallmarks of sys- strain. Unlike their wild-type counterparts, anti-dsDNA B cells in http://www.jimmunol.org/ temic lupus erythematosus (1). Mouse strains with muta- VH3H9 BALB-lpr/lpr and gld/gld mice entered B cell follicles, T 3 tions in the genes for Fas and Fas ligand (FasL) (i.e., lpr and gld) and by 10 wk of age their Abs were detectable in the serum. Strik- (2, 3) produce autoantibodies similar to those found in systemic ingly, as early as 5–6 wk of age, lpr/lpr mice had an increased lupus erythematosus patients. lpr and gld mice also develop frequency and altered localization of dendritic cells (DCs), and this lymphadenopathy, increased total serum Ig, accelerated mortality, was associated with a global increase in CD4 T cell activation. and severe nephritis, although the latter is strain dependent (1, Nevertheless, other aspects of autoimmunity, e.g., nephritis, ap- 4–15). While these ﬁndings implicate Fas/FasL deﬁciency in au- pear restricted to the MRL-lpr/lpr mouse strain. toantibody production, it is uncertain how these mutations lead to

the differentiation of autoreactive B cells. by guest on October 2, 2021 We have used the VH3H9 IgH transgene (Tg) model to track Materials and Methods anti-dsDNA B cells in both nonautoimmune-prone (BALB/c) and Experimental mice autoimmune-prone (MRL-lpr/lpr) mice in vivo (16–19). In VH3H9 BALB/c mice, anti-dsDNA B cells are present with a developmen- TgϪ and VH3H9 BALB-lpr/lpr mice were generated by breeding VH3H9 tally arrested phenotype and localize to the interface between the MRL-lpr/lpr mice with BALB/c mice, followed by backcrossing onto the T and B cell areas in the splenic white pulp. Anti-dsDNA Abs are not BALB/c background (at least seven times), and then intercrossing to generate homozygous mice. Because it was possible that MRL genes linked to produced in these mice. In contrast, anti-dsDNA B cells in VH3H9 the lpr gene on chromosome 19 (2) might be carried onto the BALB/c MRL-lpr/lpr mice have a mature phenotype, populate the splenic B background, we took a second approach to studying Fas/FasL deficiency by cell follicles, and produce anti-dsDNA Abs. also examining BALB-gld/gld mice. The gld defect, located on chromo- The purpose of the present study was to identify the specific some 1, was originally found as a spontaneous mutation in C3H mice (3, effects of Fas/FasL mutations on anti-dsDNA B cells, independent 24, 25). VH3H9 BALB/c mice were mated with BALB-gld/gld mice, fol- of the autoimmune MRL background (4, 9, 11, 20–23). To this lowed by intercrosses to generate homozygous gld/gld mice. BALB/c mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). MRL- lpr/lpr, MRLϩ/ϩ, and BALB-gld/gld (Cpt substrain, backcrossed onto the *Wistar Institute, Philadelphia, PA 19104; and †Department of Medicine, University BALB background for at least 15 generations) mice were purchased from of Pennsylvania School of Medicine, Philadelphia, PA 19104 The Jackson Laboratory (Bar Harbor, ME). VH3H9 mice have been previously described (16). The VH3H9 Tg has been backcrossed onto the Received for publication April 11, 2001. Accepted for publication June 5, 2001. BALB/c and MRL backgrounds for at least 17 generations. The costs of publication of this article were defrayed in part by the payment of page All mice were bred and maintained in a specific pathogen-free room at charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the Wistar Institute animal facility. In all experiments, mice were age matched, and BALB/c mice and TgϪ littermates were used as controls. 1 M.L.F. is supported by a Howard Hughes Medical Institute predoctoral fellowship Male and female mice were used with no apparent differences. The pres- grant and by a Gina Finzi Memorial Student Summer Fellowship from the Lupus Foundation of America; M.P.M. is supported by National Institutes of Health Grant ence of the VH3H9 Tg and the lpr and gld mutations was determined by DK33694; and J.E. is supported by National Institutes of Health Grant 5R01 PCR amplification of tail DNA with primers specific for VH3H9 (16–19), AI32137-10, the Lupus Foundation of America, and the Arthritis Foundation. Fas (26), and FasL (a gift of M. Maldonado, Department of Rheumatology, 2 Address correspondence and reprint requests to Dr. Jan Erikson, Wistar Institute, University of Pennsylvania). The primers used for FasL were as follows: Room 276, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: FasL wild-type locus, 5Ј-CTC TGA TCA ATT TTG AGG AAT CTA [email protected] AGA CGT-3Ј; FasL mutant locus, 5Ј-CTC TTG GCC ATT TAA CAT Ј 3 Abbreviations used in this paper: FasL, Fas ligand; ANA, anti-nuclear Ab; AP, CAG ACA GTT CTT-3 . The PCR conditions used for FasL were: 92°C alkaline phosphatase; DC, dendritic cell; PALS, periarteriolar lymphoid sheath; Tg, for 5 min; 92°C for 30 s, 60°C for 1 min, and 72°C for 45 s; for 36 cycles; transgene, transgenic. ending with an extension period at 72°C for 10 min.

Identification of anti-dsDNA B cells malin (Fisher Scientific, Pittsburgh, PA), then embedded in paraffin. Kid- neys were sectioned to 4-␮m thickness and stained with H&E. The ␭ The VH3H9 IgH chain paired with the V 1 L chain produces an anti- presence of renal pathology was determined as described (30, 32) by a Ͼ ␭ϩ dsDNA Ab (27, 28). The majority (average 95%) of Ig B cells in single investigator (M. P. Madaio) without knowledge of age or genotype ␭ ϩ VH3H9 BALB-lpr/lpr and BALB-gld/gld mice were Ig 1 (data not of the mice. Kidneys were graded for severity of disease in three areas shown), as was demonstrated previously for VH3H9 BALB/c and MRL- (vascular, interstitial, and glomerular) in a range of 0 (for no pathology ␭ ϩ lpr/lpr mice by flow cytometry (17, 18). Therefore, we used pan-anti-Ig evident) to 4 (most severe pathology, end stage disease) (33), and cumu- ␭ as well as anti-Ig 1 reagents to identify anti-dsDNA B cells in VH3H9 lative scores were determined. mice. In VH3H9 BALB-lpr/lpr and BALB-gld/gld mice at the time points studied (6–12 wk), Ͼ95% of the B cells in the mouse expressed surface IgM only, not IgD (data not shown), consistent with the exclusive use of Statistics the IgM-only VH3H9 Tg. Statistical significance was determined using an unpaired nonparametric Flow cytometric analysis (Wilcoxon or Mann-Whitney) test, Student’s t test, or alternate Welch t Ϫ test, when appropriate. Statistical significance was ascribed when p values Spleens were removed from VH3H9 Tg and Tg mice. Single cell sus- were less than 0.05. pensions were prepared, and erythrocytes were lysed (RBC lysing buffer; Sigma, St. Louis, MO). Cells (ϳ1 ϫ 106) were surface stained according to published protocols (29). The following Abs and secondary reagents were used: 1D3 FITC or biotin (anti-CD19), 7G6 FITC (CR2/CR1, anti- Results CD21/35), Cy34.1 FITC or biotin (anti-CD22), B3B4 FITC (anti-CD23), Lymphoproliferation in BALB-lpr/lpr mice IM7 FITC (anti-CD44), RA3-6B2 FITC or PE (anti-B220), R11-153 biotin (anti-V␭1), 145-2C11 FITC or PE (anti-CD3), GK1.5 FITC or PE (anti- BALB-lpr/lpr mice were bred to dissect the effects of Fas independent of the MRL background. As was the case for other

CD4), 53-6.72 PE (anti-CD8), H1.2F3 FITC (anti-CD69, VEA), 7D4 FITC Downloaded from or PE (anti-IL-2R␣-chain p55, CD25), HC3 FITC or PE (anti-CD11c), strains of mice onto which the lpr mutation has been bred 16-10A1 FITC (anti-B7.1/CD80), GL1 PE (anti-B7.2/CD86), 2G9 FITC (MRL, C57BL/6, C3H, and AKR) (4, 6, 7, 9–15), by 9–12 wk (anti-I-Ad/I-Ed), and anti-CD40 FITC (Becton Dickinson/PharMingen, San Diego, CA); 33D1 FITC (Leinco Technologies, St. Louis, MO); JC5.1 PE of age BALB-lpr/lpr mice had a gross increase in spleen weight (anti-V␭ total, gift of J. Kearney, University of Alabama, Birmingham, (Table I). This was largely due to significant increases in num- AL); GK1.5 (anti-CD4) and RA3-6B2 (anti-B220), which were grown as bers of B220ϩCD3ϩCD4ϪCD8Ϫ double-negative T cells and supernatants and then biotinylated; and streptavidin Red 670 (Life Tech- B220ϩCD4ϩ T cells as compared with BALB/c control mice. nologies, Gaithersburg, MD). For CD80 or CD86 staining, FcR were first http://www.jimmunol.org/ blocked by incubation with 2.4G2. Cell size was gauged by the forward scatter values of the cells. Samples were collected on a FACScan flow Altered localization and cell surface phenotype of anti-dsDNA B cytometer (Becton Dickinson, San Jose, CA) and analyzed using CellQuest software. For B and T cell analyses, 40,000–80,000 events, gated for live cells in BALB-lpr/lpr and BALB-gld/gld mice lymphocytes based on forward and side scatter, were collected for each We have previously used VH3H9 IgH Tg mice to study the reg- sample. For DC analyses, 100,000 events, gated for larger size and gran- ularity, were collected for each sample. To determine absolute numbers ulation of anti-dsDNA B cells (16, 27, 34). In vivo, B cells utilize within a cell type, their frequency within live gated splenocytes was mul- the VH3H9 IgH in combination with a variety of L chains, thus tiplied by the total number of live splenocytes (determined by trypan blue generating a heterogeneous B cell population that includes both exclusion). anti-DNA and non-DNA B cells (27). The pairing of the VH3H9 by guest on October 2, 2021 ␭ Spleen preparations for DC experiments IgH with the V 1 L chain generates an anti-dsDNA Ab (27, 28). This facilitates tracking of anti-dsDNA B cells in vivo in a diverse Spleens were injected with 0.5 ml collagenase solution (100 U/ml Liberase repertoire by the use of anti-Ig␭ reagents (17–19). Cl (Boehringer Mannheim, Indianapolis, IN) containing 0.2 mg/ml DNase I (Sigma) in HBSS (Cellgro, Herndon, VA)). Spleens were teased into Anti-dsDNA B cells in VH3H9 BALB-lpr/lpr and BALB-gld/ small fragments and then incubated at 37°C for 30 min in 400 U/ml Lib- gld mice localized in splenic B cell follicles, in contrast to their erase Cl in HBSS. Splenic fragments were then pushed through a cell Fas-sufficient counterparts, which were follicularly excluded (Fig. strainer (100 ␮M). The single cell suspension was mixed with RPMI ϩ 5 1). Strikingly, this altered localization of anti-dsDNA B cells in mM EDTA (Life Technologies) to inhibit the collagenase. Cells were then lpr/lpr and gld/gld mice was apparent at an early age (5–6 wk) and treated to lyse erythrocytes and prepared for flow cytometry, as described above. persisted in the oldest mice examined (12 wk). In all VH3H9 mouse genotypes examined, the anti-dsDNA B Anti-nuclear Ab (ANA) and Crithidia luciliae assays cells had decreased surface levels of Ig and CD21/35 (Fig. 2A), a The presence of ANAs and anti-dsDNA Abs in the serum was detected phenotype that we and others have attributed to continual Ag en- using the ANA and C. luciliae assays, respectively, as previously described counter (17, 18, 30, 35–42). At all ages examined, anti-dsDNA B ϩ (18, 30). Binding was detected using either anti-IgM IgG FITC (togeth- cells from VH3H9 BALB-lpr/lpr mice had increased levels of er, to detect total Ig) or anti-Ig␭ FITC (all from Southern Biotechnology Associates, Birmingham, AL). B220, as compared with Fas-sufficient VH3H9 BALB/c mice (Fig. 2B); these levels were similar to those in VH3H9 MRL-lpr/lpr Tissue immunohistochemistry mice (18). In terms of the other maturation/activation markers Spleens from experimental mice were prepared and stained as previously studied (CD22, CD44, and cell size), the anti-dsDNA B cells from described (18, 31). The following Abs were used: Cy34.1 FITC or biotin VH3H9 BALB-lpr/lpr mice fell into two groups that segregated (anti-CD22), RA3-6B2 biotin (anti-B220), GK1.5 FITC or biotin (anti- with age (Fig. 2C). Anti-dsDNA B cells from young (Յ8-wk) CD4), HC3 FITC (anti-CD11c), M1/70 FITC (anti-CD11b, Mac-1) VH3H9 BALB-lpr/lpr mice were similar to those in Fas-sufficient (PharMingen), and/or anti-Ig␭ alkaline phosphatase (AP; Southern Bio- technology Associates). FITC- and biotin-conjugated reagents were de- VH3H9 BALB/c mice, expressing low levels of CD22 and in- tected with the secondary reagents anti-FITC AP (Sigma) or anti-FITC creased levels of CD44 and cell size. However, in older VH3H9 HRP (Chemicon, Temecula, CA), and streptavidin AP or HRP, respec- BALB-lpr/lpr mice, (Ն8 wk of age), the anti-dsDNA B cells up- tively (Southern Biotechnology Associates). AP and HRP were developed regulated CD22, down-regulated CD44, and were smaller. This with the substrates Fast-Blue BB base (blue) (Sigma), and 3-amino-9- ethyl-carbazole (red), respectively. latter phenotype is like that seen in VH3H9 MRL-lpr/lpr mice of all ages (18). The phenotypic patterns observed in young and old Kidney pathology VH3H9 BALB-gld/gld mice were comparable with those in age- Sixteen- to twenty-four-week-old TgϪ BALB/c, BALB-lpr/lpr, and MRL- matched VH3H9 BALB-lpr/lpr mice (data not shown, n ϭ 6atՅ8 lpr/lpr mice were sacrificed, and kidneys were fixed in 10% buffered For- wk; n ϭ 7atՆ8 wk). 2372 ALTERED ANTI-dsDNA B CELL AND DC LOCALIZATION IN lpr/gld MICE

Table I. Spleen characteristics from TgϪ BALB/c, BALB-lpr/lpr, and MRL-lpr/lpr micea

6–8wk 9–12 wk

MRL-lpr/lpr BALB-lpr/lpr BALB/c MRL-lpr/lpr BALB-lpr/lpr BALB/c

Spleen weight (mg) 144.84 Ϯ 71.21 (10) 169.13 Ϯ 26.82 (7) 117.60 Ϯ 20.44 (5) 344.95 Ϯ 218.00 (6) 262.08 Ϯ 71.42 (9) 113.37 Ϯ 14.33 (6) B cells (ϫ107) 2.43 Ϯ 1.20 (8) 7.95 Ϯ 0.63 (5) 3.74 Ϯ 0.74 (3) 4.32 Ϯ 1.74 (4) 7.53 Ϯ 2.95 (7) 4.35 Ϯ 0.82 (6) CD4 T cells (ϫ107) 1.15 Ϯ 1.07 (10) 2.00 Ϯ 0.52 (7) 1.21 Ϯ 0.53 (5) 1.49 Ϯ 1.04 (6) 2.14 Ϯ 0.79 (9) 1.24 Ϯ 0.34 (6) DN T cells (ϫ106) 2.13 Ϯ 6.07 (8) 3.25 Ϯ 1.24 (5) 0.71 Ϯ 0.50 (3) 10.91 Ϯ 15.93 (4) 14.72 Ϯ 10.67 (7) 0.67 Ϯ 0.23 (6) B220ϩCD4ϩ T cells 1.35 Ϯ 1.56 (10) 3.64 Ϯ 1.35 (7) 0.79 Ϯ 0.33 (5) 3.33 Ϯ 2.67 (6) 4.04 Ϯ 1.90 (9) 0.99 Ϯ 0.40 (6) (ϫ106)

a Mice were studied over a range of ages (6–12 wk) and divided into two groups, (6–8 and 9–12 wk). Mean Ϯ SD for each strain are shown. Numbers in parentheses, number of mice examined. B cells were defined as CD19ϩ or B220ϩCD3Ϫ, CD4 T cells were defined as CD4ϩCD3ϩ, and DN T cells were defined as B220ϩCD3ϩCD4Ϫ (all lymphoid-gated cells). BALB-lpr/lpr mice (9–12 wk old) had increased spleen weight ( p ϭ 0.0004), increased number of DN T cells ( p ϭ 0.0012), and increased number of B220ϩCD4ϩ T cells ( p ϭ 0.0008) over control BALB/c mice. Compared with their TgϪ counterparts, B220ϩCD4ϩ T cell numbers were elevated 2.3-fold in 9- to 12-wk-old VH3H9 MRL-lpr/lpr mice ( p ϭ 0.0256); B cell numbers were decreased 2.2-fold in 6- to 8-wk-old VH3H9 BALB-lpr/lpr mice ( p ϭ 0.0010) and 1.6-fold in 9- to 12-wk-old VH3H9 BALB/c mice ( p ϭ 0.0173); and CD4 T cell numbers were elevated 2.4-fold in 9- to 12-wk-old VH3H9 MRL-lpr/lpr mice ( p ϭ 0.0047) and 1.4-fold in 9- to 12-wk-old VH3H9 BALB/c mice ( p ϭ 0.0303).

ϩ/ϩ Autoantibody production in lpr/lpr mice BALB-lpr/lpr mice compared with MRL and BALB/c mice Downloaded from By 10 wk of age, 100% of BALB-lpr/lpr mice produced ANAs (Fig. 4). As previously reported (49, 51–54), splenic DCs were with patterns indistinguishable from those of MRL-lpr/lpr mice clustered primarily in the bridging channels to the red pulp in (Fig. 3). BALB-gld/gld mice also developed ANAs with similar Fas-sufficient mice (Fig. 4). In contrast, DCs were spread through- patterns and frequencies (n ϭ 8, data not shown). ANA titers in out the T cell zone (periarteriolar lymphoid sheath, PALS) in BALB-lpr/lpr and MRL-lpr/lpr mice were not significantly differ- spleens of Fas/FasL-deficient mice. This altered localization was ent, and at 10 wk of age, the titer and incidence of ANAs were not evident in lpr/lpr mice as early as 5–6 wk of age and persisted up http://www.jimmunol.org/ affected by the presence of the VH3H9 Tg (Table II). Sera from to 12 wk of age, the latest time point examined. BALB-gld/gld ϭ older (9- to 12-wk-old) ANAϩ Fas/FasL-deficient BALB/c mice mice also exhibited this altered DC localization (n 3, data not contained Ig␭ϩ anti-dsDNA Abs (for VH3H9 BALB-lpr/lpr mice, shown). Furthermore, the lpr mutation resulted in an increased n ϭ 10/10; for VH3H9 BALB-gld/gld mice, n ϭ 8/8, data not frequency and number of DCs in both BALB/c and MRL strains shown). Consistent with the serum data, staining of spleens from (Fig. 5, A and B). Additionally, older lpr/lpr mice had an increased 9- to 12-wk-old Fas/FasL-deficient VH3H9 mice revealed darkly absolute number of DCs compared with younger lpr/lpr mice. staining Ig␭ϩ cells in the T cell area as well as in the bridging channels to the red pulp, which coincided with staining for synde- Unaltered DC subtypes and maturation/activation status in by guest on October 2, 2021 can-1, a marker of Ab-forming cells (data not shown and (18, 43)). lpr/lpr mice DCs in murine secondary lymphoid organs have been classified Altered DC localization and increased frequency/numbers in into two main subtypes: myeloid and lymphoid (51, 55–59). These lpr/lpr mice two DC types have been proposed to take opposing roles in T cell DCs express Fas, although its role on these cells is controversial activation and tolerance, although this remains controversial (60– (44–50). Whereas several studies have illustrated Fas-mediated 64). Myeloid DCs are marked by expression of CD11c, CD11b, apoptosis of DCs (45–47), recent data suggest not only that DCs and 33D1, and within the spleen resting myeloid DCs are concen- resist Fas-induced death, but also that signals through Fas may trated at the bridging channels to the red pulp (51, 56). These DCs induce DC maturation (50). To evaluate DCs in Fas/FasL-deficient enter the PALS upon activation and terminal maturation (49, 65, mice, spleen sections were stained with Abs to the integrin CD11c 66). Lymphoid DCs are identified by expression of CD11c, (51). Immunohistochemical staining demonstrated striking differ- NLDC145 (DEC-205), and the CD8␣-␣ homodimer, and within ences in the splenic localization of DCs in TgϪ MRL-lpr/lpr and the spleen they are normally found in the PALS (51, 52, 57). We

FIGURE 1. Altered localization of anti-dsDNA B cells in lpr/lpr and gld/gld mice. Spleen sections from VH3H9 BALB/c, VH3H9 BALB-lpr/lpr, and VH3H9 BALB-gld/gld mice were stained with Abs to Ig␭ (blue), and either CD22 or CD4 (red). Anti-dsDNA B cells localize at the T/B interface in VH3H9 BALB/c mice, but populate B cell follicles in VH3H9 BALB-lpr/lpr and BALB-gld/gld mice. n Ն 8 mice for each genotype. The Journal of Immunology 2373

FIGURE 2. The phenotype of anti-dsDNA B cells in VH3H9 BALB-lpr/lpr or BALB- gld/gld mice. Splenocytes were stained with Abs against CD19, Ig␭, CD21/35, B220, CD22, and CD44. Histograms are gated on the Ig␭Ϫ B cell population from a TgϪ BALB/c mouse (thin black line) and Ig␭ϩ cells in each mouse (gray line). Shown are representative phenotypes of Ig␭ϩ B cells in TgϪ BALB/c (5–12 wk (17, 18) and n ϭ 7, this study), VH3H9 BALB/c (5–12 wk (17, 18) and n ϭ 7, this study), VH3H9 BALB- lpr/lpr mice (5–8 wk, n ϭ 8), and VH3H9 BALB-lpr/lpr mice (8–12 wk, n ϭ 13). The underlaid histograms (thin black line) were scaled down to allow for comparison with the Ig␭ϩ cells, which only comprise ϳ10% of the B cell population in VH3H9 mice. A, Anti-dsDNA B cells in VH3H9 mice have decreased levels of Ig and CD21/35. Nota-

bly, older (8- to 12-wk) VH3H9 BALB- Downloaded from lpr/lpr mice have a reduced frequency and number of CD19ϩIg␭ϩ cells compared with younger (5–8 wk) VH3H9 BALB-lpr/lpr mice: 2.79% Ϯ 1.22 vs 6.90% Ϯ 0.96, p Ͻ 0.0001; absolute number 2.76 ϫ 106 vs 4.74 ϫ 106, p ϭ 0.0049. B, Anti-dsDNA B cells from VH3H9 BALB-lpr/lpr mice have http://www.jimmunol.org/ increased levels of B220, in contrast to VH3H9 BALB/c mice. C, Anti-dsDNA B cells from young (Ͻ8-wk) VH3H9 BALB- lpr/lpr mice are similar to VH3H9 BALB/c mice in terms of CD22 and CD44 expression, and cell size. However, in older VH3H9 BALB-lpr/lpr mice, Ͼ8 wk of age, anti-dsDNA B cells have up-regulated CD22 and down-regulated CD44, and decreased in cell size. Fifty percent of the VH3H9 BALB- by guest on October 2, 2021 lpr/lpr mice studied at 8 wk fall into each group. Ig␭ϩ B cell phenotypes in TgϪ and VH3H9 BALB/c mice are age independent (up to 12 wk); thus, they were not divided into age groups. examined the subtype of the DCs that located in the PALS of in MRL-lpr/lpr mice compared with MRLϩ/ϩ mice (data not lpr/lpr mice. Immunohistological staining with CD11b established shown, n ϭ 4 and 3, respectively). While these differences in lo- that myeloid DCs represent the majority of DCs in the PALS of calization of myeloid DCs were seen, the percentages of DCs clas- BALB-lpr/lpr mice, whereas most myeloid DCs were at the bridg- siﬁed as myeloid were statistically equivalent within backgrounds, ing channels in BALB/c mice (Fig. 6). The same pattern was seen regardless of the presence of functional Fas (Table III). The majority of CD11cϩ DCs in both TgϪ BALB-lpr/lpr and BALB/c mice were of the myeloid phenotype. In MRL-lpr/lpr and

Table II. Mean ANA titers in BALB-lpr/lpr and MRL-lpr/lpr mice are not statistically differenta

MRL-lpr/lpr BALB-lpr/lpr

10 wk VH3H9 1.80 ϫ 103 (4) 1.60 ϫ 103 (5 ) TgϪ 1.80 ϫ 103 (4) 1.00 ϫ 103 (5 )

20 wk FIGURE 3. Mice deﬁcient in Fas/FasL develop lupus autoantibodies. Combined Tgϩ and TgϪ 6.55 ϫ 103 (6) 2.70 ϫ 103 (10) Sera were tested for the presence of total ANAs by immunoﬂuorescence. Shown are representative nuclear staining patterns seen in the ANA assay a At 10 wk of age, p ϭ 0.95; at 20 wk of age, p ϭ 0.63. ANA titers were Ϫ Ͼ determined by staining with anti-IgM plus anti-IgG Abs. The VH3H9 Tg does not of Tg MRL-lpr/lpr and BALB-lpr/lpr mice. At 10 wk of age, 95% of ϭ ϭ Ϫ ϭ affect ANA production (at 10 wk, p 1.0 for MRL-lpr/lpr mice and p 0.83 for Tg MRL-lpr/lpr mice (Ref. 18 and n 5, this study) and 100% of BALB- BALB-lpr/lpr mice). Numbers in parentheses, n for each strain. BALB/c mice 10 wk lpr/lpr mice (n ϭ 12) are serum ANA positive. old have ANA titers of Ͻ10 (28). 2374 ALTERED ANTI-dsDNA B CELL AND DC LOCALIZATION IN lpr/gld MICE

FIGURE 4. DCs in lpr/lpr mice have an altered localization compared with those in Fas-sufﬁcient mice. Spleen sections from TgϪ BALB/c, BALB-lpr/lpr, MRLϩ/ϩ, and MRL-lpr/lpr mice were stained with Abs to CD11c (blue) and CD22 (red). While CD11cϩ DCs are concentrated in the bridging channels in Fas-sufﬁ- cient mice (arrows), they are spread throughout the PALS in lpr/lpr mice. This altered localization is observed at ages 5–12 wk. n Ն 6 mice for each genotype. Downloaded from

MRLϩ/ϩ mice, the majority of DCs were also of the myeloid phe- Since myeloid DCs were accumulating in the PALS of lpr/lpr notype; however, the frequency of myeloid DCs was signiﬁcantly mice, we tested whether these DCs were activated. This was not less than in the BALB/c strains. The reason for the strain difference the case. Ex vivo splenic DCs from TgϪ BALB-lpr/lpr and MRL- is unclear. lpr/lpr mice expressed levels of CD80 (B7.1), CD86 (B7.2), CD40, and MHC class II that were equivalent to those found on http://www.jimmunol.org/ DCs from nonimmunized BALB/c mice (Fig. 7 and data not shown).

T cell activation in lpr/lpr mice Consistent with previous reports (8, 33, 67, 68), the number of activated T cells in lpr/lpr mice was increased compared with BALB/c mice (Fig. 8). The B220ϪCD4ϩ T cell population in both

MRL-lpr/lpr and BALB-lpr/lpr mice had signiﬁcantly elevated by guest on October 2, 2021 percentages that were CD25ϩ and/or CD69ϩ in the spleen compared with BALB/c mice (Fig. 8B). In BALB-lpr/lpr mice, the percentages of CD4 T cells that up-regulated CD69 and CD25 were similar. However, in MRL-lpr/lpr mice, the proportion of CD4 T cells expressing CD69 was higher than the proportion expressing CD25. Interestingly, CD69ϩCD4 T cells have been isolated from the synovial ﬂuid and membrane of chronic rheumatoid

FIGURE 5. DC frequency and numbers are elevated in lpr/lpr mice. Each circle represents one mouse. E, Mice age 5–9 wk; F, mice age 10–15 wk. A, Frequency was assessed as percentage of B220ϪCD11cϩ cells of live splenocytes. Over the entire age range (5–15 wk), mean frequencies are: BALB/c, 0.95%; BALB-lpr/lpr, 1.45%; MRLϩ/ϩ, 0.68%; and MRL-lpr/lpr, 1.11%. The lpr mutant mice had significantly increased frequencies of DCs over Fas-sufficient mice. B, Mean values for the absolute number of DCs over the entire FIGURE 6. Myeloid DCs are spread throughout the PALS in BALB- age range (5–15 wk) are: BALB/c, 1.09 ϫ 106; BALB-lpr/lpr, 6.16 ϫ 106; lpr/lpr mice. Spleen sections from TgϪ BALB/c and BALB-lpr/lpr mice MRLϩ/ϩ, 9.78 ϫ 105; and MRL-lpr/lpr, 2.27 ϫ 106. The lpr mutant mice had were stained with Abs to CD22 (red) and either CD11c (top) or CD11b significantly increased numbers of DCs over Fas-sufficient mice. Statistically (bottom) (blue). Myeloid DCs are indicated by their CD11c and CD11b significant p values are shown on the graph. coexpression. n Ͼ 3 for BALB/c and BALB-lpr/lpr mice. The Journal of Immunology 2375

Table III. Frequencies of myeloid-type (33D1ϩ) and lymphoid-type (CD8␣ϩ) DCs (gated on B220ϪCD11cϩ) in Fas-sufﬁcient and lpr/lpr micea

BALB/c BALB-lpr/lpr MRLϩ/ϩ MRL-lpr/lpr

33D1ϩ 64.87 Ϯ 3.04% 63.38 Ϯ 13.40% 44.46 Ϯ 9.58% 43.43 Ϯ 14.62% CD8␣ϩ 21.11 Ϯ 9.60% 19.67 Ϯ 7.36% 29.31 Ϯ 3.34% 31.06 Ϯ 13.07% n 55Ն610

a The lpr mutation did not affect the frequencies of the subtypes as determined by flow cytometry: for BALB/c compared to BALB-lpr/lpr, p ϭ 0.8203 for myeloid-type DCs and p ϭ 0.7978 for lymphoid-type DCs; for MRLϩ/ϩ compared with MRL- lpr/lpr, p ϭ 0.8676 for myeloid-type DCs and p ϭ 0.6961 for lymphoid-type DCs. However, there was a lower frequency of myeloid type in the MRL strain than in the BALB/c strain: for BALB/c compared with MRLϩ/ϩ, p ϭ 0.0026; for BALB-lpr/lpr compared with MRL-lpr/lpr, p ϭ 0.0299. In all strains the myeloid and lymphoid subtypes do not account for 100% of the B220ϪCD11cϩ cells; other myeloid cell types do express CD11c, and there may be other DC subsets as yet undefined. The frequencies of lymphoid-type DCs was not significantly changed with strain: for BALB/c compared with MRLϩ/ϩ, p ϭ 0.1428; for BALB-lpr/lpr compared with MRL-lpr/lpr, p ϭ 0.0521. arthritis patients (69), and strikingly, these included an unusual basal expression levels of CD80, CD86, CD40, and MHC class II. CD69ϩ/CD25Ϫ T cell subset (70). CD4 T cell activation was ap- Given that DCs express Fas (44), one potential explanation for parent in the youngest animals examined (6 wk) and did not show their unique localization in lpr/lpr and gld/gld mice is that they a significant increase as the mice aged up to 12 wk. In MRL-lpr/lpr escape death from Fas-induced apoptosis and thus accumulate in Downloaded from and BALB-lpr/lpr mice, a significant fraction of the CD4ϩ T cells the splenic T cell areas. Supporting this idea, there are an increased that also expressed B220 had up-regulated CD69, but not CD25 frequency and number of DCs in lpr/lpr mice. In this regard, it is (Fig. 8C). The VH3H9 Tg did not alter the state of T cell activation intriguing that a defect in another apoptosis-related gene, caspase in the mice examined (n ϭ 6, VH3H9 BALB/c; n ϭ 7, VH3H9 10, resulted in an accumulation of DCs in the T cell zone of the MRL-lpr/lpr; n ϭ 12, VH3H9 BALB-lpr/lpr, data not shown). lymph node of one patient with autoimmune lymphoproliferative syndrome type II (71). Another possibility arises from the recent Nephritis in Fas/FasL-deficient BALB/c mice suggestion that Fas is not a death receptor for DCs, but rather acts http://www.jimmunol.org/ Despite the presence of high titer ANAs, BALB-lpr/lpr mice de- to induce their maturation (50). Therefore, the phenotype we have veloped minimal histological evidence of nephritis (Fig. 9). The documented in this work could result from defective maturation/ degree of nephritis was unaffected by the VH3H9 Tg (data not activation due to the absence of Fas. Experiments are underway to shown; for VH3H9 BALB/c, n ϭ 4; VH3H9 MRL-lpr/lpr, n ϭ 4; distinguish between these two possibilities. and VH3H9 BALB-lpr/lpr, n ϭ 4). Furthermore, other signs of In agreement with previous publications (8, 33, 67, 68), CD4 T autoimmune disease, such as failure to groom and skin lesions, cell activation was apparent in the youngest lpr/lpr animals exam- evident in MRL-lpr/lpr mice by 12–16 wk, were not apparent in ined (6 wk). The changes in the DCs and T cells in lpr/lpr mice Ͼ BALB-lpr/lpr or BALB-gld/gld mice up to 24 wk of age (n 10 most likely feed back upon one another as DC-T cell cross-talk by guest on October 2, 2021 for each, data not shown). occurs. For instance, activated T cells may influence DC localization in lpr/lpr mice by secreting DC chemoattractants (reviewed in Discussion Ref. 72) in the PALS. Furthermore, since DCs can potentially kill lpr or gld mutations bred onto nonautoimmune-prone strains of T cells via FasL (73), the absence of Fas/FasL interactions between mice demonstrate that Fas/FasL deficiency results in autoantibody T cells and DCs most likely contributes to the presence of acti- production and lymphoproliferation (1, 4–13). We extend these vated T cells in lpr/lpr mice. studies by documenting that Fas/FasL deficiency, alone, is suffi- Finally, BALB-lpr/lpr mice, like other lpr/lpr and gld/gld cient for anti-dsDNA B cells to bypass follicular exclusion and strains (4, 9–11, 13), did not develop severe nephritis. The BALB- produce autoantibodies. Furthermore, this is accompanied by an lpr/lpr mice are unique, however, in that they produce autoanti- alteration in the localization of DCs that is evident as early as 5 wk bodies that appear indistinguishable from those in MRL-lpr/lpr of age and persists into adulthood. mice. It is notable that CD4ϩ T cells from MRLϩ/ϩ mice are DCs in the spleens of MRL-lpr/lpr, BALB-lpr/lpr, and BALB- hyperresponsive (23). Furthermore, older MRLϩ/ϩ mice develop gld/gld mice were spread throughout the PALS. This localization ANAs and nephritis (4, 9, 11) consistent with the hypothesis that was due to an influx of myeloid DCs, which are typically found in other factors contribute to the full-blown nephritis phenotype. For the PALS after activation. In this case, however, the localization example, MRL-lpr/lpr mice may produce autoantibodies that more was not associated with DC maturation/activation, as indicated by readily form immune deposits or are more likely to elicit an

FIGURE 7. Maturation/activation status of DCs ex vivo. Spleen cells were stained with Abs to B220, CD11c, and either CD80, CD86, CD40, or I-Ad. Histograms (thin black lines) were gated on splenic B220ϪCD11cϩ cells taken ex vivo. The maturation/activation phenotype of DCs from TgϪ BALB/c and BALB-lpr/lpr spleens looks similar. n Ͼ 4 mice for each genotype. For comparison, the top row of histograms (BALB/c) is overlaid (gray line) with staining of activated DCs from BALB/c spleens that have been cultured for6hin1␮g/ml LPS (n ϭ 3), which resulted in up-regulation of the markers shown here. 2376 ALTERED ANTI-dsDNA B CELL AND DC LOCALIZATION IN lpr/gld MICE

FIGURE 8. T cell activation in 10-wk-old Fas/ FasL-deficient mice. Spleen cells were stained with Abs to B220, CD4, and either CD25 or CD69. Num- bers in the histograms are means Ϯ SD. A, Dot plots were gated on B220ϪCD4ϩ cells and B220ϩCD4ϩ cells. B, Both MRL-lpr/lpr (n ϭ 15) and BALB-lpr/lpr (n ϭ 12) mice have more CD4 T cells that are CD69ϩ and/or CD25ϩ compared with BALB/c mice (n ϭ 9) (for CD25, p Ͻ 0.0001 for both strains; for CD69, p Ͻ 0.0001 for both strains). In BALB-lpr/lpr mice, percentages of CD4ϩ T cells that were CD69ϩ or CD25ϩ were not significantly different (p ϭ 0.45); however, in MRL-lpr/lpr mice, the proportion of CD4 T cells expressing CD69 is significantly higher than the proportion expressing CD25 (p ϭ 0.0011). C, A significant Downloaded from proportion of B220ϩCD4ϩ cells in MRL-lpr/lpr (n ϭ 4) and BALB-lpr/lpr (n ϭ 6) mice expresses CD69, but not CD25 (compared with BALB/c mice, p ϭ 0.0381 and 0.0260, respectively; for CD69 compared with CD25, p ϭ 0.0286 and 0.0022, respectively). Twenty percent of the few B220ϩCD4ϩ cells present in

BALB/c mice (n ϭ 6) express both CD69 and CD25. http://www.jimmunol.org/

inflammatory response. Alternatively, the renal inflammatory re- mice, are resistant to damage induced by a vigorous immune re- by guest on October 2, 2021 sponse to deposited Ab may be more vigorous in MRL mice. sponse to peroral infection with Toxoplasma gondii, even though BALB/c mice, on the other hand, may be protected from kidney both strains produce IFN-␥ in the response (78). pathology by virtue of their Th2-like nature (74–77). In this vein, In summary, we have shown that Fas/FasL deficiency, on the it has been demonstrated that BALB/c mice, but not C57BL/6 nonautoimmune-prone BALB/c background, results in several alterations in anti-dsDNA B cells. As early as 6 wk of age, they populate the splenic B cell follicle, and by 10–12 wk of age show signs of terminal differentiation into Ab-forming cells. In addition, 6-wk-old lpr/lpr mice, compared with wild-type mice, have an altered localization and increased frequency of CD11cϩ DCs as well as a significant amount of activated T cells. We hypothesize that alterations in the DCs, as well as a lack of FasL-mediated T cell killing by DCs, may lead to the presence of activated, autoreactive CD4 T cells, which are critical for autoantibody production (79–81).

Acknowledgments We thank Ryan G. Fields, Kathryn M. Potts, and Jodi L. Buckler for critical reading of the manuscript, and Dr. Laura Mandik-Nayak for initiating the BALB-lpr/lpr studies.

References FIGURE 9. Nephritis in TgϪ lpr/lpr mice. Kidneys from mice at 16–24 1. Tan, E. M. 1989. Antinuclear antibodies: diagnostic markers for autoimmune wk of age were scored for glomerulonephritis (0–4ϩ), interstitial nephritis diseases and probes for cell biology. Adv. Immunol. 44:93. ϩ ϩ 2. Watanabe-Fukunaga, R., C. I. Brannan, N. G. Copeland, N. A. Jenkins, and (0–4 ), and vasculitis (0–4 ). Each circle represents the cumulative score S. Nagata. 1992. Lymphoproliferation disorder in mice explained by defects in for one mouse. The lines represent the mean values for that mouse strain. Fas antigen that mediates apoptosis. Nature 356:314. ,Indicates that MRL-lpr/lpr mice (mean score 10.36) developed signiﬁ- 3. Takahashi, T., M. Tanaka, C. I. Brannan, N. A. Jenkins, N. G. Copeland, T. Suda ,ء cantly greater nephritis than either BALB/c (mean score 2.62; p ϭ 0.0061) and S. Nagata. 1994. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76:969. or BALB-lpr/lpr (mean score 3.20; p Ͻ 0.0001) mice. Disease in the 4. Izui, S., V. E. Kelley, K. Masuda, H. Yoshida, J. B. Roths, and E. D. Murphy. BALB-lpr/lpr mice was not signiﬁcantly different from that in BALB/c 1984. Induction of various autoantibodies by mutant gene lpr in several strains of mice (p ϭ 0.81). mice. J. Immunol. 133:227. The Journal of Immunology 2377

5. Cohen, P. L., and R. A. Eisenberg. 1991. Lpr and gld: single gene models of 35. Raff, M. C., J. J. Owen, M. D. Cooper, A. R. Lawton, M. Megson, and systemic autoimmunity and lymphoproliferative disease. Annu. Rev. Immunol. W. E. Gathings. 1975. Differences in susceptibility of mature and immature 9:243. mouse B lymphocytes to anti-immunoglobulin-induced immunoglobulin sup- 6. Sidman, C. L., J. D. Marshall, and H. Von Boehmer. 1992. Transgenic T cell pression in vitro: possible implications for B-cell tolerance to self. J. Exp. Med. receptor interactions in the lymphoproliferative and autoimmune syndromes of 142:1052. lpr and gld mutant mice. Eur. J. Immunol. 22:499. 36. Sidman, C. L., and E. R. Unanue. 1975. Receptor-mediated inactivation of early 7. Davidson, W. F., F. J. Dumont, H. G. Bedigian, B. J. Fowlkes, and H. C. Morse. B lymphocytes. Nature 257:149. 1986. Phenotypic, functional, and molecular genetic comparisons of the abnormal 37. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, lymphoid cells of C3H-lpr/lpr and C3H-gld/gld mice. J. Immunol. 136:4075. R. A. Brink, H. Pritchard-Briscoe, J. S. Wothersponn, R. H. Loblay, K. Raphael, 8. Davidson, W. F., C. Calkins, A. Hugins, T. Giese, and K. L. Holmes. 1991. et al. 1988. Altered immunoglobulin expression and functional silencing of self- Cytokine secretion by C3H-lpr and -gld T cells: hypersecretion of IFN-␥ and reactive B lymphocytes in transgenic mice. Nature 334:676. tumor necrosis factor-␣ by stimulated CD4ϩ T cells. J. Immunol. 146:4138. 38. Hartley, S. B., M. P. Cooke, D. A. Fulcher, A. W. Harris, S. Cory, A. Basten, and 9. Pisetsky, D. S., S. A. Caster, J. B. Roths, and E. D. Murphy. 1982. lpr gene C. C. Goodnow. 1993. Elimination of self-reactive B lymphocytes proceeds in control of the anti-DNA antibody response. J. Immunol. 128:2322. two stages: arrested development and cell death. Cell 72:325. 10. Warren, R. W., S. A. Caster, J. B. Roths, E. D. Murphy, and D. S. Pisetsky. 1984. 39. Cooke, M. P., A. W. Heath, K. M. Shokat, Y. Zeng, F. D. Finkelman, The influence of the lpr gene on B cell activation: differential antibody expression P. S. Linsley, M. Howard, and C. C. Goodnow. 1994. Immunoglobulin signal in lpr congenic mouse strains. Clin. Immunol. Immunopathol. 31:65. transduction guides the specificity of B cell-T cell interactions and is blocked in 11. Kelley, V. E., and J. B. Roths. 1985. Interaction of mutant lpr gene with back- tolerant self-reactive B cells. J. Exp. Med. 179:425. ground strain influences renal disease. Clin. Immunol. Immunopathol. 37:220. 40. Takahashi, K., Y. Kozono, T. J. Waldschmidt, D. Berthiaume, R. J. Quigg, 12. Gillette-Ferguson, I., and C. L. Sidman. 1994. A specific intercellular pathway of A. Baron, and V. M. Holers. 1997. Mouse complement receptors type 1 (CR1; apoptotic cell death is defective in the mature peripheral T cells of autoimmune CD35) and type 2 (CR2; CD21): expression on normal B cell subpopulations and lpr and gld mice. Eur. J. Immunol. 24:1181. decreased levels during the development of autoimmunity in MRL/lpr mice. 13. Wloch, M. K., A. L. Alexander, A. M. M. Pippen, D. S. Pisetsky, and J. Immunol. 159:1557. G. S. Gilkeson. 1996. Differences in V␬ gene utilization and VH CDR3 sequence 41. Nguyen, K.-A. T., L. Mandik, A. Bui, J. Kavaler, A. Norvell, J. G. Monroe, among anti-DNA from C3H-lpr mice and lupus mice with nephritis. Eur. J. Im- J. H. Roark, and J. Erikson. 1997. Characterization of anti-single-stranded DNA

munol. 26:2225. B cells in a non-autoimmune background. J. Immunol. 159:2633. Downloaded from 14. Giese, T., and W. F. Davidson. 1995. In CD8ϩ T cell-deficient lpr/lpr mice, 42. Roark, J. H., A. Bui, K.-A. Nguyen, L. Mandik, and J. Erikson. 1997. Persistence CD4ϩB220ϩ and CD4ϩB220Ϫ T cells replace B220ϩ double-negative T cells as of functionally compromised anti-dsDNA B cells in the periphery of non-auto- the predominant populations in enlarged lymph nodes. J. Immunol. 154:4986. immune mice. Int. Immunol. 9:1615. 15. Nagata, S., and T. Suda. 1995. Fas and fas ligand: lpr and gld mutations. Immu- 43. Smith, K. G. C., T. D. Hewitson, G. J. V. Nossal, and D. M. Tarlinton. 1996. The nol. Today 16:39. phenotype and fate of the antibody-forming cells of the splenic foci. Eur. J. Im- 16. Erikson, J., M. Z. Radic, S. A. Camper, R. R. Hardy, C. Carmack, and munol. 26:444. M. Weigert. 1991. Expression of anti-DNA immunoglobulin transgenes in non- 44. Ashany, D., A. Savir, N. Bhardwaj, and K. B. Elkon. 1999. Dendritic cells are autoimmune mice. Nature 349:331. resistant to apoptosis through the Fas (CD95/APO-1) pathway. J. Immunol. 163: 17. Mandik-Nayak, L., A. Bui, H. Noorchashm, A. Eaton, and J. Erikson. 1997. 5303. http://www.jimmunol.org/ Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: local- 45. Matsue, H., D. Edelbaum, A. C. Hartmann, A. Morita, P. R. Bergstresser, ization to the T-B interface of the splenic follicle. J. Exp. Med. 186:1257. H. Yagita, K. Okumura, and A. Takashima. 1999. Dendritic cells undergo rapid 18. Mandik-Nayak, L., S.-j. Seo, C. Sokol, K. M. Potts, A. Bui, and J. Erikson. 1999. apoptosis in vitro during antigen-specific interaction with CD4ϩ T cells. J. Im- MRL-lpr/lpr mice exhibit a defect in maintaining developmental arrest and fol- munol. 162:5287. licular exclusion of anti-double-stranded DNA B cells. J. Exp. Med. 189:1799. 46. Kawamura, T., M. Azuma, N. Kayagaki, S. Shimada, H. Yagita, and 19. Mandik-Nayak, L., S.-j. Seo, A. Eaton-Bassiri, D. Allman, R. R. Hardy, and K. Okumura. 1999. Fas/Fas ligand-mediated elimination of antigen-bearing J. Erikson. 2000. Functional consequences of the developmental arrest and fol- Langerhans cells in draining lymph nodes. Br. J. Dermatol. 141:201. licular exclusion of anti-double-stranded DNA B cells. J. Immunol. 164:1161. 47. Servet-Delprat, C., P.-O. Vidalain, O. Azocar, F. Le Deist, A. Fischer, and 20. Vidal, S., D. H. Kono, and A. N. Theofilopoulos. 1998. Loci predisposing to C. Rabourdin-Combe. 2000. Consequences of Fas-mediated human dendritic cell autoimmunity in MRL-Faslpr and C57BL/6-Faslpr mice. J. Clin. Invest. 101:696. apoptosis induced by measles virus. J. Virol. 74:4387. 21. Watson, M., J. Rao, G. Gilkeson, P. Ruiz, E. Eicher, D. Pisetsky, A. Matsuzawa, 48. Willems, F., Z. Amraoui, N. Vanderheyde, V. Verhasselt, E. Aksoy, C. Scaffidi, by guest on October 2, 2021 J. Rochelle, and M. Seldin. 1992. Genetic analysis of MRL-lpr mice: relationship M. Peter, P. Krammer, and M. Goldman. 2000. Expression of c-FLIPL and re- of the Fas apoptosis gene to disease manifestations and renal disease-modifying sistance to CD95-mediated apoptosis of monocyte-derived dendritic cells: inhi- loci. J. Exp. Med. 176:1645. bition by bisindolylmaleimide. Blood 95:3478. 22. Vyse, T. J., and B. L. Kotzin. 1998. Genetic susceptibility to systemic lupus 49. DeSmedt, T., B. Pajak, E. Muraille, L. Lespagnard, E. Heinen, P. D. Baetselier, erythematosus. Annu. Rev. Immunol. 16:261. J. Urbain, O. Leo, and M. Moser. 1996. Regulation of dendritic cell numbers and 23. Vratsanos, G. S., S. Jung, Y.-M. Park, and J. Craft. 2001. CD4ϩ T cells from maturation by lipopolysaccharide in vivo. J. Exp. Med. 184:1413. lupus-prone mice are hyperresponsive to T cell receptor engagement with low 50. Rescigno, M., V. Piguet, B. Valzasina, S. Lens, R. Zubler, L. French, V. Kindler, and high affinity peptide antigens: a model to explain spontaneous T cell activa- J. Tschopp, and P. Ricciardi-Castagnoli. 2000. Fas engagement induces the mat- tion in lupus. J. Exp. Med. 193:329. uration of dendritic cells (DCs), the release of interleukin (IL)-1␤, and the pro- 24. Davidson, W. F., K. L. Holmes, J. B. Roths, and H. C. Morse. 1985. Immunologic duction of interferon␥ in the absence of IL-12 during DC-T cell cognate inter- abnormalities of mice bearing the gld mutation suggest a common pathway for action: a new role for Fas ligand in inflammatory responses. J. Exp. Med. 192: murine nonmalignant lymphoproliferative disorders with autoimmunity. Proc. 1661. Natl. Acad. Sci. USA 82:1219. 51. Steinman, R. M., M. Pack, and K. Inaba. 1997. Dendritic cells in the T-cell areas 25. Roths, J. B., E. D. Murphy, and E. M. Eicher. 1984. A new mutation, gld, that of lymphoid organs. Immunol. Rev. 156:25. produces lymphoproliferation and autoimmunity in C3H/HeJ. J. Exp. Med. 52. Kamath, A. T., J. Pooley, M. A. O’Keeffe, D. Vremec, Y. Zhan, A. M. Lew, A. 159:1. D’Amico, L. Wu, D. F. Tough, and K. Shortman. 2000. The development, mat- 26. Liu, T. T., B. Hilliard, E. B. Samoilova, and Y. Chen. 2000. Differential roles of uration, and turnover rate of mouse spleen dendritic cell populations. J. Immunol. Fas ligand in spontaneous and actively induced autoimmune encephalomyelitis. 165:6762. Clin. Immunol. 95:203. 53. Wu, Q., Y. Wang, J. Wang, E. O. Hedgeman, J. L. Browning, and Y.-X. Fu. 1999. 27. Radic, M. Z., M. A. Mascelli, J. Erikson, H. Shan, and M. Weigert. 1991. Ig H The requirement of membrane lymphotoxin for the presence of dendritic cells in and L chain contributions to autoimmune specificities. J. Immunol. 146:176. lymphoid tissues. J. Exp. Med. 190:629. 28. Roark, J. H., C. L. Kuntz, K.-A. Nguyen, A. J. Caton, and J. Erikson. 1995. 54. Sousa, C. R. e., S. Hieny, T. Scharton-Kersten, D. Jankovic, H. Charest, Breakdown of B cell tolerance in a mouse model of SLE. J. Exp. Med. 181:1157. R. N. Germain, and A. Sher. 1997. In vivo microbial stimulation induces rapid 29. Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, and K. Hayakawa. 1991. CD40 ligand-independent production of interleukin 12 by dendritic cells and their Resolution and characterization of pro-B and pre-B cell stages in normal mouse redistribution to T cell areas. J. Exp. Med. 186:1819. bone marrow. J. Exp. Med. 173:1213. 55. Vremec, D., and K. Shortman. 1997. Dendritic cell subtypes in mouse lymphoid 30. Eaton-Bassiri, A. S., L. Mandik-Nayak, S. Seo, M. P. Madaio, M. P. Cancro, and organs: cross-correlation of surface markers, changes with incubation, and dif- J. Erikson. 2000. Alterations in splenic architecture and the localization of anti- ferences among thymus, spleen, and lymph nodes. J. Immunol. 159:565. double stranded DNA B cells in aged mice. Int. Immunol. 12:915. 56. Pulendran, B., J. Lingappa, M. K. Kennedy, J. Smith, M. Teepe, A. Rudensky, 31. Jacob, J., R. Kassir, and G. Kelsoe. 1991. In situ studies of the primary immune C. R. Maliszewski, and E. Maraskovsky. 1997. Developmental pathways of den- response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of dritic cells in vivo: distinct function, phenotype, and localization of dendritic cell responding cell populations. J. Exp. Med. 173:1165. subsets in FLT3 ligand-treated mice. J. Immunol. 159:2222. 32. Chan, O., M. P. Madaio, and M. J. Shlomchik. 1997. The roles of B cells in 57. Groth, B. F. d. S. 1998. The evolution of self-tolerance: a new cell arises to meet MRL/lpr murine lupus. Ann. NY Acad. Sci. 815:75. the challenge of self-reactivity. Immunol. Today 19:448. 33. Chan, O. T., L. G. Hannum, A. M. Haberman, M. P. Madaio, and 58. Grabbe, S., E. Kampgen, and G. Schuler. 2000. Dendritic cells: multi-lineal and M. J. Shlomchik. 1999. A novel mouse with B cells but lacking serum antibody multi-functional. Immunol. Today 21:431. reveals an antibody-independent role for B cells in murine lupus. J. Exp. Med. 59. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of 189:1639. immunity. Nature 392:245. 34. Roark, J. H., C. L. Kuntz, K.-A. Nguyen, L. Mandik, M. Cattermole, and 60. Smith, A. L., and B. F. d. S. Groth. 1999. Antigen-pulsed CD8␣ϩ dendritic cells J. Erikson. 1995. B cell selection and allelic exclusion of an anti-DNA immu- generate an immune response after subcutaneous injection without homing to the noglobulin transgene in MRL-lpr/lpr mice. J. Immunol. 154:4444. draining lymph node. J. Exp. Med. 189:593. 2378 ALTERED ANTI-dsDNA B CELL AND DC LOCALIZATION IN lpr/gld MICE

61. Haan, J. M. M. d., S. M. Lehar, and M. J. Bevan. 2000. CD8ϩ but not CD8Ϫ 71. Wang, J., L. Zheng, A. Lobito, F. K.-M. Chan, J. Dale, M. Sneller, X. Yao, dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192:1685. J. M. Puck, S. E. Straus, and M. J. Leonardo. 1999. Inherited human caspase 10 62. Sousa, C. R. e., G. Yap, O. Schultz, N. Rogers, M. Schito, J. Aliberti, S. Hieny, mutations underlie defective lymphocyte and dendritic cell apoptosis in autoim- and A. Sher. 1999. Paralysis of dendritic cell IL-12 production by microbial mune lymphoproliferative syndrome type II. Cell 98:47. products prevents infection-induced immunopathology. Immunity 11:637. 72. Sallusto, F., and A. Lanzavecchia. 1999. Mobilizing dendritic cells for tolerance, 63. Maldanado-Lopez, R., T. DeSmedt, P. Michael, J. Godfroid, B. Pajak, priming, and chronic inflammation. J. Exp. Med. 189:611. C. Heirman, K. Thielemans, O. Leo, J. Urbain, and M. Moser. 1999. CD8␣ϩ and ϩ Ϫ 73. Suss, G., and K. Shortman. 1996. A subclass of dendritic cells kills CD4 T cells CD8␣ subclasses of dendritic cells direct the development of distinct T helper via Fas/Fas ligand-induced apoptosis. J. Exp. Med. 183:1789. cells in vivo. J. Exp. Med. 189:587. 74. Hsieh, C. S., S. E. Macatonia, A. O’Garra, and K. M. Murphy. 1995. T cell 64. Maldonado-Lopez, R., T. DeSmedt, B. Pajak, C. Heirman, K. Thielemans, genetic background determines default T helper phenotype development in vitro. ␣ϩ O. Leo, J. Urbain, C. R. Maliszewski, and M. Moser. 1999. Role of CD8 and J. Exp. Med. 181:713. ␣Ϫ CD8 dendritic cells in the induction of primary immune responses in vivo. 75. Reiner, S. L., and R. M. Locksley. 1995. The regulation of immunity to Leish- J. Leukocyte Biol. 66:242. mania major. Annu. Rev. Immunol. 13:151. 65. DeSmedt, T., B. Pajak, G. G. B. Klaus, R. J. Noelle, J. Urbain, O. Leo, and 76. Louis, J., H. Himmelrich, C. Parra-Lopez, F. Tacchini-Cottier, and P. Launois. M. Moser. 1998. Cutting edge: antigen specific T lymphocytes regulate lipopo- 1998. Regulation of protective immunity against Leishmania major in mice. lysaccharide-induced apoptosis of dendritic cells in vivo. J. Immunol. 161:4476. Curr. Opin. Immunol. 10:459. 66. Sousa, C. R. e., and R. N. Germain. 1999. Analysis of adjuvant function by direct 77. Louis, J. A., F. Conceicao, H. Himmelrich, F. Tacchini-Cottier, and P. Launois. visualization of antigen presentation in vivo: endotoxin promotes accumulation of ϩ antigen-bearing cells in the T cell areas of lymphoid tissue. J. Immunol. 162: 1998. Anti-Leishmania effector functions of CD4 Th1 cells and early events 6552. instructing Th2 development and susceptibility to Leishmania major in BALB/c 67. Giese, T., and W. F. Davidson. 1992. Evidence for early onset, polyclonal acti- mice. Adv. Exp. Med. Biol. 452:53. vation of T cell subsets in mice homozygous for lpr. J. Immunol. 149:3097. 78. Liesenfeld, O., J. Kosek, J. S. Remington, and Y. Suzuki. 1996. Association of ϩ ␥ 68. Chan, O., and M. J. Shlomchik. 1998. A new role for B cells in systemic auto- CD4 T cell-dependent, interferon- -mediated necrosis of the small intestine immunity: B cells promote spontaneous T cell activation in MRL-lpr/lpr mice. with genetic susceptibility of mice to peroral infection with Toxoplasma gondii. J. Exp. Med. 184:597. J. Immunol. 160:51. ϩ 69. Laffon, A., R. Garcua-Vicuna, A. Humbria, A. A. Postigo, A. L. Corbi, 79. Santoro, T. J., J. P. Portanova, and B. L. Kotzin. 1988. The contribution of L3T4 Downloaded from M. O. DeLandazuri, and F. Sanchez-Madrid. 1991. Up-regulated expression and T cells to lymphoproliferation and autoantibody production in MRL-lpr/lpr mice. function of VLA-4 fibronectin receptors on human activated T cells in rheuma- J. Exp. Med. 167:1713. toid arthritis. J. Clin. Invest. 88:546. 80. Merino, R., M. Iwamoto, L. Fossati, and S. Izui. 1993. Polyclonal B cell activa- ϫ 70. Afeltra, A., M. Galeazzi, G. D. Sebastiani, G. M. Ferri, D. Caccavo, tion arises from different mechanisms in lupus-prone (NZB NZW)F1 and M. A. Addessi, R. Marcolongo, and L. Bonomo. 1997. Coexpression of CD69 MRL/MpJ-lpr/lpr mice. J. Immunol. 151:6509. and HLADR activation markers on synovial fluid T lymphocytes of patients 81. Koh, D.-R., A. Ho, A. Rahemtulla, W.-P. Fung-Leung, H. Griesser, and T.-W. affected by rheumatoid arthritis: a three-color cytometric analysis. Int. J. Exp. Mak. 1995. Murine lupus in MRL/lpr mice lacking CD4 or CD8 T cells. Eur. Pathol. 78:331. J. Immunol. 25:2558. http://www.jimmunol.org/ by guest on October 2, 2021