Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Psychoneuroimmunology wikipedia , lookup
Adaptive immune system wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Molecular mimicry wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Innate immune system wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Genetic Dissection of Sle Pathogenesis: Sle3 on Murine Chromosome 7 Impacts T Cell Activation, Differentiation, and Cell Death This information is current as of June 18, 2017. Chandra Mohan, Ying Yu, Laurence Morel, Ping Yang and Edward K. Wakeland J Immunol 1999; 162:6492-6502; ; http://www.jimmunol.org/content/162/11/6492 Subscription Permissions Email Alerts This article cites 85 articles, 43 of which you can access for free at: http://www.jimmunol.org/content/162/11/6492.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 References Genetic Dissection of Sle Pathogenesis: Sle3 on Murine Chromosome 7 Impacts T Cell Activation, Differentiation, and Cell Death1 Chandra Mohan,2* Ying Yu,† Laurence Morel,† Ping Yang,† and Edward K. Wakeland2* A rich series of immunological events underlies the pathogenesis of lupus (1–3). Both generalized B cell hyperactivity and T cell-driven B cell autoantibody production have been well documented in murine and human lupus. Likewise, generalized T cell abnormalities, as well as help rendered by clonally expanded Ag-specific Th cells, are both likely to contribute to pathology, as discussed below. These different immunological aberrations ultimately lead to the formation of pathogenic antinuclear Abs (ANAs),3 the hallmark of this disease. Although the genetic basis of these mechanisms has long remained a mystery, recent murine studies are just beginning to reveal their intricacies. Susceptibility to lupus in murine models has been mapped to several loci, as recently reviewed (4 – 6). Our studies have focused on the NZM2410 strain, derived from a cross between New Zealand Black (NZB) and New Zealand White (NZW) mice (7). This lupus-prone strain demonstrates splenomegaly, a significant expansion of activated CD41 T cells and B cells, high-titered ANAs, and early-onset immune-complex glomerulonephritis (GN) (7, 8). To map these immunological traits, a (NZM2410 3 C57BL/6 *Simmons Arthritis Research Center and Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235; and †Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610 Received for publication October 22, 1998. Accepted for publication March 9, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants PO1 AI39824 and RO1 AR44894 and by a grant from the National Arthritis Foundation. 2 Address correspondence and reprint requests to Drs. Chandra Mohan and Edward K. Wakeland, Simmons Arthritis Research Center and the Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235-8884. E-mail addresses: [email protected] and [email protected] 3 Abbreviations used in this paper: ANA, anti-nuclear Ab; NZB, New Zealand Black; NZW, New Zealand White; GN, glomerulonephritis; B6, C57BL/6; PI, propidium iodide; AICD, activation-induced cell death; LN, lymph node; TTP, tristetraprolin. Copyright © 1999 by The American Association of Immunologists (B6)) 3 NZM2410 backcross analysis was undertaken several years ago (9). That mapping study revealed that genomic intervals on chromosomes 1 (Sle1), 4 (Sle2), 7 (Sle3), and 17 (Sle4) were strongly linked to lupus nephritis. Of relevance to this study, the locus on chromosome 7 (Sle3) showed significant linkage to GN, with peak linkage at the pink eye-dilution ( p) locus (X 2 5 16.7; p , 1024; logarithm of the odds score 5 4). Other independent mapping studies have also mapped lupus susceptibility to murine chromosome 7, close to Sle3. Using an (NZB 3 NZW)F2 mapping panel, Kono et al. (10) mapped a lupus susceptibility locus, Lbw5, in the vicinity of Sle3. Subsequently, the same workers mapped susceptibility to lymphadenopathy and anti-dsDNA production in another disease model, using an (MRL.Faslpr 3 B6.Faslpr)F2 analysis, to Lmb3, again in the vicinity of Sle3 (11). Using an (MRL/ lpr 3 CAST/Ei) backcross panel, Watson et al. (12) mapped susceptibility to lupus nephritis to a more centromeric locus on murine chromosome 7, close to ckmm. Thus, this genomic interval on murine chromosome 7 appears to harbor a gene(s) that potentially dictates some critical pathogenic mechanisms leading to lupus in several different disease-prone strains. Deciphering the relative immunopathological contributions of these loci to disease has been greatly facilitated by the generation and characterization of congenic mice bearing these individual lupus susceptibility intervals (4, 13–17). Adopting this powerful approach, we have recently shown that Sle1 triggers loss of tolerance to chromatin, apparently without any generalized abnormalities in lymphocyte activation, differentiation, or apoptosis (15, 17). In contrast, Sle2 leads to generalized B cell hyperactivity, elevated levels of splenic and peritoneal B1a cells, and increased serum polyclonal/polyspecific IgM, but with no evidence of IgG ANAs, T cell defects, or GN (15, 16). This study focuses on B6.NZMc7 mice, which are B6 mice rendered congenic for the NZM2410derived Sle3 interval, as diagrammed in Fig. 1. Our initial studies with this congenic strain had revealed increased serum levels of polyclonal/polyspecific IgM and IgG Abs, accompanied by an 0022-1767/99/$02.00 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Polyclonal, generalized T cell defects, as well as Ag-specific Th clones, are likely to contribute to pathology in murine lupus, but the genetic bases for these mechanisms remain unknown. Mapping studies indicate that loci on chromosomes 1 (Sle1), 4 (Sle2), 7 (Sle3), and 17 (Sle4) confer disease susceptibility in the NZM2410 lupus strain. B6.NZMc7 mice are C57BL/6 (B6) mice congenic for the NZM2410-derived chromosome 7 susceptibility interval, bearing Sle3. Compared with B6 controls, B6.NZMc7 mice exhibit elevated CD4:CD8 ratios (2.0 vs 1.34 in 1- to 3-mo-old spleens); an age-dependent accumulation of activated CD41 T cells (33.4% vs 21.9% in 9- to 12-mo-old spleens); a more diffuse splenic architecture; and a stronger immune response to T-dependent, but not T-independent, Ags. In vitro, Sle3-bearing T cells show stronger proliferation, increased expansion of CD41 T cells, and reduced apoptosis (with or without anti-Fas) following stimulation with anti-CD3. With age, the B cells in this strain acquire an activated phenotype. Thus, the NZM2410 allele of Sle3 appears to impact generalized T cell activation, and this may be causally related to the low grade, polyclonal serum autoantibodies seen in this strain. Epistatic interactions with other loci may be required to transform this relatively benign phenotype into overt autoimmunity, as seen in the NZM2410 strain. The Journal of Immunology, 1999, 162: 6492– 6502. The Journal of Immunology 6493 FIGURE 1. The NZM2410 strain spontaneously develops lupus. In a backcross study involving this strain, three non-MHC genomic intervals showed significant linkage to autoantibody production and/or GN: Sle1 on chromosome 1, Sle2 on chromosome 4, and Sle3 on chromosome 7 (9). In the latter study, peak linkage for Sle3 was noted at the p locus (28 cM). By repeated backcrossing and microsatellite marker-based selection, the 95% confidence interval flanking Sle3 on NZM2410 chromosome 7 has been bred onto the B6 background (14). The introgressed chromosome 7 interval encompassing Sle3 is shown in white, with termini at D7 MIT56 and D7 MIT62. Lbw5 (10) and Lmb3 (11) are lupus susceptibility loci mapped by other groups to the same genomic interval. Materials and Methods Mice B6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and subsequently bred in our animal colony. The derivation of B6 congenic mice bearing NZM2410-derived lupus susceptibility intervals has been detailed previously (14). As depicted in Fig. 1, B6.NZMc7 mice are B6 mice congenic (homozygotes) for a 40-cM interval on murine chromosome 7, encompassing the 95% confidence interval flanking Sle3, with termini at D7 MIT56 and D7 MIT62. The entire congenic interval is derived from the NZW parent of the NZM2410 strain. All mice used for this study were bred and housed in a conventional colony, under identical conditions, at the University of Florida Department of Animal Resources. As no sex differences were noted in the expression of any of the phenotypes described, both male and female mice were used in this study. Nevertheless, for the in vivo antigenic challenge experiments, female mice were used. Cell preparation Splenocytes were depleted of RBCs using 0.83% NH4Cl, and single-cell suspensions were prepared. Total splenic B cells were prepared as described before (17, 18), using pretitrated amounts of anti-Thy-1 (Accurate Chemicals, New York, NY) and rabbit complement (Accurate Chemicals) to lyse T cells, and were typically 85–95% pure. Splenic T cells were prepared as described (17, 18). Briefly, red cell-depleted splenocytes were loaded onto nylon wool columns (Robbins Scientific, Sunnyvale, CA) and incubated at 37°C for 45 min. Nonadherent cells were washed through and then incubated with pretitrated amounts of anti-I-Ab (clone K25– 8.7, Accurate Chemicals) and anti-CD24 (PharMingen, San Diego, CA) on ice for 45 min. Ab-bound cells were lysed with rabbit complement (Accurate Chemicals), yielding T cells with .90% purity. Bone marrow cells were obtained by flushing femurs and tibia with medium. Peritoneal cavity cells were obtained by injecting medium into the peritoneal cavity and extracting the cells with transfer pipettes. Prepared single-cell suspensions were counted and used for culture or flow cytometric analysis, as described below. Flow cytometric analysis, sorting, and Abs Flow cytometric analysis (FACS) was performed as described previously (16, 19). Briefly, cells were first blocked with staining medium (PBS, 5% horse serum, and 0.05% azide) containing 10% normal rabbit serum. Cells were then stained on ice with optimal amounts of FITC, PE, or biotinconjugated primary mAbs diluted in staining medium for 30 min. Follow- ing two washes, biotin-conjugated Abs were revealed using streptavidin-PE (Life Technologies, Grand Island, NY) or streptavidin-Quantum Red (Sigma, St. Louis, MO). Cell staining was analyzed using a FACScan (Becton Dickinson, San Jose, CA). Dead cells were excluded on the basis of scatter characteristics and propidium iodide (PI) uptake, and 10,000 events were acquired per sample. The following dye- or biotin-coupled Abs were obtained from PharMingen and used at pretitrated dilutions: CD4 (RM4-5), CD5 (53-7.3), CD8 (Ly-2), CD23 (B3B4), CD24 (M1/69), CD25 (7D4), CD43 (S7), CD44 (IM7), CD45R/B220 (RA3-6B2), CD62L (MEL14), CD69 (H1.2F3), CD80/B7-1 (16-10A1), and CD86/B7-2 (GL1). Cell stimulation assays F(ab9)2 goat anti-mouse IgM (Cappel, Durham, NC) and LPS (Sigma) were used for B cell stimulation at concentrations from 0.1 to 100 mg/ml. Likewise, graded doses of anti-CD3 (PharMingen), rIL-2 (Chiron Therapeutics, Emeryville, CA) or keyhole limpet hemocyanin (KLH; Calbiochem, La Jolla, CA) were added to assess T cell responses to stimuli. For these assays total splenocytes or splenic T cells (5 3 105/well) were cultured for 72 h in 200-ml cultures, in serum-free HL-1 medium (HyCor Biomedicals, Irvine, CA), with or without added stimuli. Lymphocyte response was assessed either by assaying the extent of proliferation, using [3H]TdR incorporation (ICN Biomedicals, Costa Mesa, CA) over the last 18 h of culture or by measuring by ELISA the amount of IL-2, IL-4, or IFN-g secreted at the indicated time points postculture. For the experiments depicted in Fig. 4, 5 3 105 splenocytes per well were cultured with anti-CD3 and anti-CD28 (1 mg/ml each, PharMingen). At 24, 48, and 72 h postculture, the absolute numbers of CD41 and CD81 T cells in culture were determined by counting live (i.e., PI-excluding) cells and by FACS analysis. For assaying activation-induced cell death (AICD), total splenocytes were stimulated with anti-CD3 and anti-CD28 for 48 h. Then, blasts (consisting of .90% T cells) were purified over Ficoll, washed, and recultured with plate-bound anti-CD3, with or without antiFas (2 mg/ml, clone Jo2, PharMingen) or control hamster IgG (PharMingen). After the indicated culture periods, the percentage of apoptosis was determined, as described below. Cell death assays Purified splenic B cells or T cells, or T cell blasts, prepared as detailed above, were cultured, with or without added stimuli. Aliquots of this culture were assayed at 0, 24, 48, and 72 h for the percentage of apoptotic cells in culture, using hypotonic lysis and PI incorporation, as described before (20). Briefly, the cell pellets were resuspended in hypotonic PI solution (3.4 mM sodium citrate, 50 mg/ml PI, 0.1% Triton X-100, 1 mM Tris, and 0.1 mM EDTA) and analyzed by FACS, using logarithmic scales. Apoptotic nuclei were distinguished by their hypodiploid DNA content and scatter characteristics, after excluding the debri. Cell death was also assayed by monitoring annexin binding, as described (21). Annexin is known to bind to phosphatidylserine exposed on the outer leaflets of cells undergoing apoptosis. For assaying this, lymphocytes or T cell blasts were stained with Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 18% incidence of severe (i.e., .50% of glomeruli affected) immune complex-mediated GN (15). By comparing the cellular phenotypes seen in the B6.NZMc7 strain with those of age-matched B6 controls, the present study aims to understand the pathogenic mechanisms through which Sle3 might contribute to disease. 6494 Sle3 IMPACTS T CELL ACTIVATION AND APOPTOSIS PE- or biotin-labeled anti-CD4, anti-CD8, or anti-B220, as described above. Following a washing step, the cells were incubated with annexinFITC (PharMingen) in HEPES-containing buffer and then analyzed on a flow cytometer. IL ELISAs IL produced in culture was assayed by ELISA, as described previously (17). The following reagents were purchased from PharMingen: anti-IL-2; anti-IL-4; anti-IFN-g; biotinylated anti-IL-2; biotinylated anti-IL-4; biotinylated anti-IFN-g; and rIL-2, rIL-4, and IFN-g standards. Briefly, Immulon I plates (Dynatech Laboratories, Chantilly, VA) precoated with the “capture” Ab (anti-IL-2, anti-IL-4, or anti-IFN-g) were blocked and then incubated for 2 h with culture supernatants (diluted 1:2) or serial dilutions of the rIL standard. Captured IL was detected using biotin-coupled anti-IL, avidin-alkaline phosphatase (1:10,000, Sigma), and p-nitrophenylphosphate substrate (Sigma). ODs were converted to pg/ml using the derived standard curve. In vivo immunization FIGURE 2. Splenic sections from B6 (top) and B6.NZMc7 (bottom) were immunostained with anti-B220 (purple) and anti-CD3 (orange). Shown sections were obtained from 9- to 12-mo-old mice and are representative of five to eight mice of each strain. Magnification, 310. Immunochemistry Immunochemistry on splenic sections was performed, as described previously (8). Briefly, fragments of spleens were snap frozen in liquid nitrogen and stored at 270°C, until time of sectioning. Cryosections 0.4 – 0.6 mm were fixed in acetone and air dried. Slides were rehydrated with TBS-0.1% BSA and then blocked with 5% normal rabbit serum and 0.3% H202/100% MeOH. T cells were tagged by sequential incubations with biotin-coupled anti-CD3 (1 mg/ml, PharMingen) and avidin-alkaline phosphatase. B cells were identified using FITC-coupled anti-B220 (5 mg/ml, PharMingen) and peroxidase-coupled anti-FITC (Boehringer Mannheim). In addition, the size and numbers of germinal centers in the spleens of the immunized mice were assessed using biotin-coupled peanut agglutinin. All incubations were performed for 30 min at room temperature in a moist chamber. Sections were developed using the peroxidase substrate diaminobenzidine (Sigma) and the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium (Sigma) and then washed and mounted using Permount (Fisher, Pittsburgh, PA). Statistics Data obtained for the B6.NZMc7 mice were compared with those from the control B6 mice using the Student’s t test. For all experiments, the mean (6SEM) values observed are shown. Results Lymphoid organs and lymphocyte subsets in B6.NZMc7 mice Sle3 did not seem to impact the development or cellular composition of the primary lymphoid organs. Thus, the marrow and thymus from B6.NZMc7 mice exhibited the appropriate distributions of B cells and T cells, respectively, at the different developmental stages (data not shown). All differences observed were confined to the secondary lymphoid organs. Although B6.NZMc7 mice did not differ from B6 controls in the size or cellularity of their spleens, the T cell and B cell zones in these mice were poorly organized, with frequent intermingling of T cell and B cell foci, compared with B6 spleens, as illustrated in Fig. 2. However, this immunohistological phenotype was a relatively late event, being apparent only in the older age group (9- to 12-mo-old mice). B6.NZMc7 spleens did not differ from B6 spleens in the numbers or size of germinal centers after immune challenge (data not shown). Table I illustrates the distribution of T cell and B cell subsets in the peripheral lymphoid organs of B6.NZMc7 mice. Because the total splenocyte numbers did not differ between the 2 strains, any observed differences in lymphocyte percentages also reflect differences in absolute numbers. The B6.NZMc7 spleens did not differ significantly from B6 controls in the numbers of B1, B2, or total T cells, at any age. However, B6.NZMc7 spleen and lymph node (LN) had significantly greater percentages and numbers of CD41 T cells relative to CD81 T cells, leading to elevated CD4:CD8 ratios compared with age-matched B6 controls (Fig. 3A; Table I). This pattern was noted at all ages tested. Thus, 1- to 3-mo-old and 4- to 6-mo-old B6.NZMc7 spleens exhibited mean splenic CD4: CD8 ratios of 2.0, and 1.77, respectively, compared with the corresponding B6 ratios of 1.34 and 1.22 ( p , 0.05 and p , 0.001, respectively). Likewise, B6.NZMc7 LN also exhibited significant increases in the absolute numbers of CD41 T cells, but not CD81 T cells, compared with B6 controls. Thus, for example, 4- to 6-moold B6.NZMc7 nodes exhibited 31.7% CD41 T cells and mean CD4:CD8 ratios of 1.07, compared with the corresponding B6 values of 23.9% and 0.82, respectively ( p , 0.01). Interestingly, the Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 B6 and B6.NZMc7 mice (four or five mice per group, age 2.5 mo) were injected i.p. with DNP-KLH (100 mg/mouse; Calbiochem) in CFA and boosted 2 weeks later with 100 mg/mouse DNP-KLH in IFA. Immunized mice were seromonitored for anti-hapten Abs for several weeks thereafter, as illustrated in Results. At sacrifice, the spleens were stained with peanut agglutinin to assess the size and numbers of germinal centers, as described below. In addition, the T cell response to the immunogen was gauged by measuring IL production (as detailed above) upon rechallenge with KLH in vitro. To assess response to thymus-independent Ags, independent groups of mice (four per group) were challenged i.p. with trinitrophenol (TNP)LPS (20 mg/mouse, Sigma) and seromonitored for anti-hapten response for 2 mo postchallenge. Briefly, serum anti-DNP and anti-TNP Abs were determined by assaying serial dilutions of the test sera on DNP-BSA-coated (Calbiochem) or TNP-BSA-coated (Accurate Chemicals) Immulon-II plates (Dynatech), respectively. Bound Abs were revealed with alkaline phosphatase-conjugated anti-mouse IgM or anti-mouse IgG (Boehringer Mannheim, Indianapolis, IN), utilizing p-nitrophenylphosphate as a substrate. The isotypes of anti-DNP Abs were determined by using isotypespecific enzyme conjugates. The avidity of these serum Abs was determined by gauging the amount of competitor (e-DNP-lysine, Sigma) that was required for reducing the ELISA reactivity to DNP-BSA by 50% (ID50). The Journal of Immunology 6495 Table I. Lymphocyte subsets in secondary lymphoid organsa Mice 1–3 Months Old B6 B6.NZMc7 4–6 Months Old 9–12 Months Old B6 B6.NZMc7 B6 B6.NZMc7 b Spleens Total cells (31026) % B cells % B1a cells % CD41 % CD81 CD4:CD8 ratio % CD691CD4 T cellsb Lymph nodes %B-cells % CD41 % CD81 CD4:CD8 ratio % CD691CD4 T cellsa 101 6 4.4 53.4 6 1.8 6.1 6 0.8 18.7 6 1.0 13.9 6 1.2 1.34 6 0.05 15.8 6 1.3 93.1 6 7.1 55.7 6 2.2 5.3 6 1.7 20.8 6 0.7* 10.3 6 0.7* 2.0 6 0.6* 20.6 6 1.6* 96.3 6 5.9 49.5 6 1.4 3.9 6 0.8 17.7 6 0.7 14.4 6 0.8 1.22 6 0.06 21.8 6 1.3 114 6 11.3 46.4 6 1.9 4.0 6 1.0 22.1 6 0.8*** 12.5 6 0.9* 1.77 6 0.13*** 23.9 6 2.5* 93.5 6 3.5 51.1 6 1.7 5.1 6 0.5 18.3 6 0.6 15.8 6 0.7 1.16 6 0.04 21.9 6 2.1 105 6 5.9 46.9 6 2.6 4.4 6 0.4 21.7 6 1.4* 13.9 6 1.2 1.56 6 0.12** 33.4 6 2.1*** 34.7 6 2.8 26.2 6 1.0 22.6 6 1.3 1.16 6 0.1 21.6 6 3.0 26.8 6 3.3 36.2 6 1.5*** 22.9 6 1.3 1.58 6 0.1* 21.3 6 2.3 35.0 6 3.3 23.9 6 0.6 29.0 6 1.1 0.82 6 0.03 27.8 6 1.4 29.6 6 4.7 31.7 6 2.1** 29.6 6 1.5 1.07 6 0.06** 26.1 6 2.6 41.2 6 3.3 18.9 6 1.4 23.3 6 1.8 0.81 6 0.07 35.9 6 3.5 23.6 6 2.6*** 23.5 6 2.2* 29.4 6 3.1 0.80 6 0.05 39.6 6 4.9 T cell:B cell ratios and the absolute numbers of T cells were significantly elevated in B6.NZMc7 LN compared with B6 controls, at all ages, as illustrated in Table I. Sle3 impacts lymphocyte activation B6.NZMc7 splenic T cells also appeared to be spontaneously activated, having increased expression of CD69, compared with B6 T cells (Table I and Fig. 3A). This phenotype became progressively stronger with age. Thus, in 9- to 12-mo-old B6.NZMc7 spleens, on the average, 33.4% of CD41 T cells expressed CD69, compared with 21.9% of B6 CD41 T cells ( p , 0.001). A similar trend was seen with the expression of two other activation markers, CD25 and CD44 (data not shown). Although splenic CD81 T cells from B6.NZMc7 mice also appeared to be more activated than their B6 counterparts, these differences were not as pronounced as those seen with CD41 T cells. In contrast, no significant differences were seen in the activation status of B6 and B6.NZMc7 LN T cells (Table I). In addition to the T cells, B6.NZMc7 B cells also appeared to be spontaneously activated. As illustrated in Fig. 3B, B6.NZMc7 splenic B cells from 9- to 12-mo-old mice exhibited significantly higher levels of surface I-Ab, CD44, and B7-2. This phenotype was late in onset, as B cells from younger B6 and B6.NZMc7 spleens did not differ from each other, by surface phenotype. At all ages tested, B6.NZMc7 spleens did not differ from B6 spleens with respect to the numbers or percentages of B1a or B1b cells. Likewise, there were no significant differences in the numbers or percentages of B1a or B1b cells in their peritoneal cavities (data not shown). We next assessed the functional status of B6.NZMc7 lymphocytes. B6.NZMc7 and B6 splenic B cells showed a similar proliferative response to anti-IgM and LPS (data not shown). However, when stimulated with anti-CD3 and anti-CD28, B6.NZMc7 T cells behaved differently. As illustrated in Fig. 4, B6.NZMc7 CD41 T cells demonstrated significantly increased expansion in culture, compared with B6 CD41 T cells. Thus, by 72 h postculture, B6.NZMc7 CD41 T cells exhibited more than a 3-fold increase in absolute numbers ( p , 0.004). A similar trend was not noted for CD81 T cells. Indeed, in all B6 cultures, there were always more CD81 T cells than CD41 T cells 72 h poststimulation, but the reverse was true with B6.NZMc7 T cells, as can be seen in Fig. 4. In both the B6 and B6.NZMc7 cultures, 72 h poststimulation, the T cells were uniformly large in size and exhibited similarly high levels of CD69 and Fas and reduced levels of L-selectin, typical of activated T cell blasts (data not shown). The increase in absolute numbers of B6.NZMc7 CD41 T cells in culture could reflect an increased production of nascent T cells and/or reduced apoptosis of activated T cells. Indeed, both appear to be contributing, as illustrated in Figs. 5 and 6. Compared with B6 T cells, B6.NZMc7 T cells showed significantly increased proliferation, as assessed by [3H]TdR incorporation, in response to stimulation with anti-CD3 (Fig. 5A), and IL-2 (Fig. 5B). These experiments were performed with 2- to 4-mo-old mice, and their splenic T cells did not differ in the surface levels of CD3 or IL-2R (data not shown). However, B6.NZMc7 T cells produced similar quantities of IL-2, IL-4, and IFN-g upon stimulation (data not shown). Sle3 impacts T cell apoptosis Apoptosis was studied by two complementary methods, hypotonic lysis with PI incorporation (20) and annexin binding (21), as detailed in Materials and Methods. In the former method, apoptotic cells were identified by their hypodiploid DNA content and light scatter characteristics. The latter method is based on the principle that dying cells expose phosphatidylserine on the outer membrane leaflets, which can be tagged by annexin binding. This method has the added advantage of allowing double or triple staining with other surface markers. This permits one to gauge cell death in selected lymphocyte populations. By both methods, B6 and B6.NZMc7 splenic B cells showed similar rates of apoptosis, with or without anti-IgM stimulation, as illustrated in Fig. 6A. Likewise, splenic T cells from both strains showed similar rates of spontaneous apoptosis in culture, with no stimuli added. In contrast, B6.NZMc7 T cells exhibited reduced Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 a The percentages of different lymphocyte subsets in the spleens and LN of 1- to 3-mo-old, 4- to 6-mo-old, and 9- to 12-mo-old B6 and B6.NZMc7 mice are tabulated. Data represent the mean (6SEM) values obtained from the indicated numbers of mice. b Numbers for spleens: for 1- to 3-mo-old mice, n 5 16 –36 for B6 and n 5 10 –28 for B6.NZMc7; for 4- to 6-mo-old mice, n 5 21–29 for B6 and n 5 8 –15 for B6.NZMc7; and for 9- to 12-mo-old mice, n 5 22–30 for B6 and n 5 14 –20 for B6.NZMc7. c Displayed percentages represent the % of CD4 T cells that expressed CD69. d Data were obtained from inguinal LN, with a similar trend being noted in other LN. B6 and B6.NZMc7 nodes did not differ in total cell numbers. e Numbers for LN: for 1- to 3-mo-old mice, n 5 24 for B6 and n 5 17 for B6.NZMc7; for 4- to 6-mo-old mice, n 5 21 for B6 and n 5 9 for B6.NZMc7; and for 9- to 12-mo-old mice, n 5 15 for B6 and n 5 8 –17 for B6.NZMc7. *p , 0.05, significantly different from B6 values; **p , 0.01, significantly different from B6 values; and ***p , 0.001, significantly different form B6 values. 6496 Sle3 IMPACTS T CELL ACTIVATION AND APOPTOSIS FIGURE 3. A, Two-dimensional plots depict the expression of CD4, CD8, and CD69 on B6 or B6.NZMc7 splenic lymphocytes, as determined by flow cytometry. Top, Individual dots in the upper right quadrants represent CD691CD41 T cells. Bottom, Percentages of cells in the lower right (i.e., CD41 T cells) and upper left (i.e., CD81 T cells) were used to derive the CD4:CD8 ratios. Shown data were obtained from 9-mo-old mice and are representative of data obtained from 14 –30 mice of each strain at this age, as detailed in Table I. B, Mean fluorescence intensity of I-Ab, CD44, and B7-2 expression on B2201 splenic B cells from 9- to 12-mo-old B6 (black bars) and B6.NZMc7 (gray bars) mice. Each bar represents the mean (6SEM) value of the mean fluorescence intensity recorded in three individual mice of each strain, stained and analyzed within the same experiment. For the shown experiment, B6.NZMc7 B cells had significantly higher expression levels of I-Ab (p , 0.02), CD44 (p , 0.05), and B7-2 (p , 0.05). These data are representative of four independent FACS staining experiments. AICD. For these experiments, splenocytes were first stimulated for 48 h with anti-CD3 and anti-CD28. Then, the lymphoblasts were Ficoll purified from the cultures, washed, and recultured on antiCD3-coated wells for the indicated durations. Importantly, the 6 h data points should accurately reflect the apoptotic rates of the plated T cell blasts, as this time lapse is too short for any cell divisions to have occurred. Importantly, the B6.NZMc7 T cell blasts consistently showed reduced apoptotic rates compared with the B6 T cell blasts, at the 6 h and 40 h time points. This was confirmed to occur with both CD41 and CD81 T cells by the annexin binding experiments (Fig. 6, B and C). Thus, both CD41 and CD81 T cell blasts showed impaired AICD, even in the presence of anti-Fas Abs. B6 and B6.NZMc7 T cells showed no consistent difference in the expression levels of Fas or FasL (data not shown). Although T cell blasts from the lupus-prone NZM2410 strain exhibited a similar phenotype (Fig. 6, B and C), they were very sensitive to Fas-mediated apoptosis. The latter observation correlates well with the heightened expression of Fas on NZM2410 blasts, compared with B6 or B6.NZMc7 T cell blasts (data not shown). Sle3 augments the immune response to antigenic challenge The immune responsiveness of these mice was assessed by challenging them with a T-dependent (DNP-KLH), or a T-independent (TNP-LPS) Ag. As illustrated in Fig. 7, upon secondary challenge with DNP-KLH, B6.NZMc7 mice showed significantly elevated levels of IgM (about 3–5 fold) and IgG (nearly two-fold) anti-DNP Abs, for several months postchallenge. The anti-DNP Abs in B6 and B6.NZMc7 mice did not differ in their avidity for the hapten (Fig. 7E) or the IgG subclass distribution (data not shown). Importantly, B6.NZMc7 T cells responded more vigorously to rechallenge with KLH by IL production (IL-2 production is depicted in Fig. 7F). However, these two strains did not differ in the relative quantities of IL-4 vs IFN-g produced upon rechallenge, suggesting that Sle3 does not impact the Th1/Th2 balance (data not shown). B6 and B6.NZMc7 immune-challenged mice did not differ in the numbers or sizes of germinal centers, as revealed by staining with peanut agglutinin. Finally, B6 and B6.NZMc7 mice showed a similar humoral response when challenged with the T-independent Ag, TNP-LPS (data not shown). Discussion End-organ damage incited by selected subsets of ANAs is likely to represent a final common pathogenic event in all models of murine lupus, as well as in human lupus. Studies performed over the past 3 decades have collectively suggested that the generation of pathogenic ANAs is not due to a single immunological aberration, but indeed represents the consequence of a multitude of underlying Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 4. For these experiments, 5 3 105 splenocytes from 2- to 4-mo-old mice were cultured with anti-CD3 and anti-CD28 (1 mg/ml each). At 0, 24, 48, and 72 h postculture, the absolute numbers of CD41 and CD81 T cells in culture were determined by enumerating viable (i.e., PIexcluding) cells in culture and by FACS analysis using anti-CD4 and antiCD8 Abs. Each data point represents the mean (6SEM) of triplicate cultures. Shown are the absolute numbers of CD41 T cells from B6 (F) and B6.NZMc7 (E), and CD81 T cells from B6 () and B6.NZMc7 mice (ƒ). In the depicted experiment, B6.NZMc7 cultures showed significantly higher numbers of CD41 T cells, compared with B6 cultures, at 48 h (p , 0.001) and 72 h (p , 0.004) postculture. This experiment is representative of five independent experiments. The Journal of Immunology pathogenic mechanisms, as reviewed in References 1–3. In particular, generalized B cell hyperactivity and T cell-driven autoantibody production by autoreactive B cells are both likely to contribute to ANA formation. Several investigators have examined the features of T cells in the various murine models of lupus, as well as in human SLE. Of relevance to this work, an expansion of activated CD41 T cells has been well documented in several lupus-prone (or genetically related) strains, including (NZB 3 NZW) F1, (NZB 3 SWR) F1, NZB.H2bm12, NZM2410, NZW, and Faslpr/lpr mice, as well as in human lupus (Refs. 22–25 and C. Mohan, unpublished observations). Although several of the mouse models are also characterized by elevated CD4:CD8 ratios, this has not been a characteristic feature of human SLE (reviewed in Ref. 25). Experiments involving knockouts, Ab-mediated blocking, and in vitro cultures have clearly demonstrated the critical role played by Th cells in driving B cell autoantibody production in murine and human lupus (26 – 32). Importantly, parallel studies have documented generalized abnormalities in T cell signal transduction (33–37) and apoptosis (38 – 43) in murine and human lupus. FIGURE 6. A, Purified B6 (black bars) or B6.NZMc7 (white bars) splenic B cells, T cells, or Ficoll-purified T cell blasts (see Materials and Methods) were cultured with or without anti-IgM (anti-u) or anti-CD3 for the indicated durations. Following this culture, the percentage of apoptotic cells in culture was assayed flow cytometrically after hypotonic lysis and PI incorporation, as detailed in Materials and Methods. Each bar represents the mean (6SEM) percentage apoptosis detected in B cells or T cells obtained from three individual mice, for each strain, or the mean of triplicate cultures of T cell blasts purified from individual B6 or B6.NZMc7 mice. The experiment shown was performed with cells obtained from 2- to 4-mo-old mice and is representative of at least three independent experiments. In the shown experiment, B6.NZMc7 T cell blasts show significantly reduced levels of apoptosis at 6 h (p , 0.004) and 40 h (p , 0.02) poststimulation. B, Alternatively, annexin binding was used to quantitate cell death in CD41 or CD81 T cells. T cell blasts were purified from B6, B6.NZMc7, or NZM2410 spleens (as detailed in Materials and Methods) and recultured for 6 h on anti-CD3-coated wells. The percentages of annexin-binding CD41 T cell blasts (which are annotated in the histograms shown) and CD81 T cell blasts were determined by flow cytometry. The shown plots are representative of data obtained from 4 –13 individual mice of each strain. C, The extent of cell death among CD41 or CD81 T cell blasts in restimulated cultures was determined by annexin binding, as demonstrated in Fig. 6B. In addition, some cultures also received anti-Fas (2 mg/ml) or control hamster Ig. Each bar represents the mean (6SEM) percentage of annexin binding seen in triplicate cultures of T cell blasts obtained from B6 (white bars), B6.NZMc7 (striped bars), or NZM2410 (cross-hatched bars) mice. In this experiment, compared with the B6 controls, significantly less annexin binding was seen among B6.NZMc7 CD41 T cell blasts (p , 0.03) and CD81 T cell blasts (p , 0.003) 6 h postrestimulation. Likewise, reduced Fas-mediated cell death was also seen in B6.NZMc7 CD41 (p , 0.0005) and CD81 (p , 0.02) T cell blasts, compared with B6 T cell blasts. The presented data are representative of three or four independent experiments. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 5. A, Splenocytes (5 3 105) from 2- to 4-mo-old B6 (black bars) or B6.NZMc7 (white bars) mice were cultured with graded doses of anti-CD3 for 72 h. The extent of lymphocyte proliferation was ascertained by [3H]TdR incorporation during the last 18 h of culture. Each bar represents the mean (6SEM) cpm incorporated by triplicate cultures. In this experiment, B6.NZMc7 T cells showed a significantly increased proliferative response to anti-CD3, compared with B6 controls, at doses of 0.1 (p , 0.04), 1 (p , 0.02), and 10 mg/ml (p , 0.01). This experiment is representative of six independent experiments. B, Purified splenic T cells (2 3 105) from 2- to 4-mo-old mice were cultured with graded doses of rIL-2 for 72 h. The extent of lymphocyte proliferation was ascertained by [3H]TdR incorporation during the last 18 h of culture. Each bar represents the mean (6SEM) cpm incorporated by triplicate cultures. In the shown experiment, B6.NZMc7 T cells showed a significantly heightened proliferative response to rIL-2, compared with B6 T cells (p , 0.02). This experiment is representative of three independent experiments. 6497 6498 Sle3 IMPACTS T CELL ACTIVATION AND APOPTOSIS Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 7. B6 or B6.NZMc7 mice 2.5 mo old (four or five mice per group) were injected i.p. with DNP-KLH (100 mg/ml) in CFA and boosted 2 weeks later. The mice were bled on days 0 (preimmune), 14 (primary response), and 21 (secondary response), and at intervals thereafter, as indicated in the figure. The experiments shown in plots A through E are representative of four independent challenge experiments. In all cases, the shown data represent the mean (6SEM) ELISA ODs in the B6 and B6.NZMc7 study groups, consisting of four or five mice each, respectively. A, The levels of IgM anti-DNP Abs in serum (diluted 1:300) were assayed at serial time points as shown. B6.NZMc7 sera exhibit significantly higher levels of IgM anti-DNP Abs than B6 sera on days 21 (p , 0.03), 98 (p , 0.01), and 128 (p , 0.003) postchallenge. B, Sera obtained on day 21 postchallenge were assayed for IgM anti-DNP Abs, using serial dilutions of the sera. C, The levels of IgG anti-DNP Abs in serum (diluted 1:2500) were assayed at serial time points, as shown. Compared with B6 controls, B6.NZMc7 sera show significantly higher IgG anti-DNP Ab levels at days 21 (p , 0.05) and 51 (p , 0.01) postchallenge. D, Sera obtained on day 21 postchallenge were assayed for IgG anti-DNP Abs, using serial dilutions of the sera. E, The avidity of the postchallenge anti-DNP Abs was gauged by determining the concentration of e-DNP-lysine (mM) that was required to inhibit serum binding to DNP-BSA on the ELISA plates by 50% (ID50). Sera from day 21 postchallenge diluted 1:2500 were used for these assays. Each bar represents the mean (6SEM) ID50 value obtained in the B6 or B6.NZMc7 study group, which each consisted of four or five mice. F, To assess the T cell response to the immunogen, splenic T cells (5 3 105/well) purified 4 mo postimmunization were stimulated with graded doses of KLH, presented by irradiated, T-depleted, syngeneic splenocytes (5 3 105/well). Shown are the triplicate mean (6SEM) levels of IL-2-produced 48-h poststimulation by T cells purified from an individual B6 or an individual B6.NZMc7 mouse immunized in the same experiment. These data are representative of data obtained from four immunized mice of each strain. The Journal of Immunology 6499 Superimposed on this generalized (i.e., polyclonal) T cell activation, a select few potentially pathogenic Th clones are likely to be further expanded in an Ag-driven fashion. This concept is supported by TCR clonotype analyses (44 – 46) and the demonstration of T cell antigenic specificities to a variety of nuclear, DNA-binding proteins, including histones, nucleosomes, ribosomal P2, and DNase I, as well as ANA-derived idiopeptides (18, 47–53). It is likely that the combined events of generalized, polyclonal T cell activation and the focused expansion of a few Ag-specific T cell clones are together responsible for driving ANA production by pathogenic B cell clones and/or end-organ disease. The present study advances an interval on murine chromosome 7 as harboring one such gene that may be responsible for the intrinsic, generalized T cell abnormality that characterizes lupus. As discussed earlier, mapping studies indicate that this genomic interval confers susceptibility to ANA and/or GN in several murine models of lupus, including the NZM2410 strain (9 –12). To elucidate the specific immunological contributions of Sle3 independent of other lupus susceptibility loci, this genomic interval has been selectively bred onto the B6 background. Analysis of these B6.NZMc7 congenic mice reveals that Sle3 leads to an increase in the absolute numbers of splenic and LN CD41 T cells and a reduction in CD81 T cells, leading to elevated CD4:CD8 ratios (Table I). Although the total numbers of splenic (CD41 and CD81) T cells are relatively similar between the 2 strains, an age-dependent increase is observed in the absolute numbers of activated CD41 T cells in B6.NZMc7 spleens. Indeed, this in vivo phenotype is recapitulated in vitro, as demonstrated by the accumulation of activated CD41 T cells in culture, upon stimulation. Both increased cell proliferation and reduced AICD appear to contribute to this accumulation. This accumulation of activated T cells over time may be causally related to the B cell surface phenotype (i.e., increased expres- sion of activation markers; Fig. 3B). Indeed, this perpetual, low grade stimulation of bystander B cells may be responsible for the low grade, polyclonal humoral autoimmunity seen in this strain (15). Finally, as one would predict, Sle3 augments the immune response to T-dependent, but not T-independent, Ags (Fig. 7). Thus far, no differences have been noted in the functional status or the subset distributions (B1a, B1b, and B2) of B cells between B6 and B6.NZMc7 mice. Ongoing experiments, utilizing bone marrow transfers and B cell- or T cell-deficient mice, are aimed at dissecting out the relative contributions of B cells, T cells, and non-lymphocytes to the observed phenotypes in this strain. It is important to note that these phenotypes are also present in the original NZM2410 strain, as well as in other lupus-prone strains, as discussed above. Indeed, in the NZM2410 strain, these phenotypes are even more pronounced (C. Mohan et al., manuscript in preparation). Also, in contrast to the lupus-prone strains, the B6.NZMc7 mice show relatively low levels of ANAs and a lower penetrance of GN (15). Additional genes that differ between the B6 and NZM2410 strain are likely to account for these differences. Indeed, it appears that in the presence of genes that actively breach tolerance to chromatin (e.g., Sle1), loci such as Sle3 can have a dramatic impact on the amplification and diversification of the incipient autoimmune response. This notion is supported by the robust humoral and cellular autoimmune phenotypes seen in B6.NZMc1/c7 bicongenic mice (Fig. 8) (90). Interestingly, induced mutations of several other T cell molecules have been shown to impact some of the same phenotypes modulated by Sle3. Following T cell activation, several molecules are known to regulate the delicate balance between resting and activated T cells. In particular, activated T cells are purged from the immune system via Fas/FasL-mediated AICD, as reviewed in Ref. 54. IL-2 plays a pivotal role in facilitating this process, in addition to its previously documented roles in T cell activation and Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 8. The lupus susceptibility interval on murine chromosome 7, harboring Sle3, appears to primarily impact T cell activation and differentiation. In the context of an otherwise normal B6 genome, Sle3 leads to spontaneous T cell activation, reduced AICD, and skewed CD4:CD8 ratios. This failure to efficiently purge activated (presumably, anti-self) T cells from the immune system may account for the various phenotypes noted in this strain, including the age-dependent accumulation of activated CD41 T cells (Table I); heightened humoral and cellular immune response to antigenic challenge (Fig. 7); spontaneous B cell activation (Fig. 3B); and polyclonal, humoral autoimmunity (15). More importantly, the T cell phenotypes impacted by Sle3 could have grave disease implications when this interval is bred onto a background that exhibits incipient autoimmunity. The B6.NZMc1 strain, congenic for Sle1, exhibits breakage in tolerance to chromatin (17). Although the latter strain exhibits anti-H2A/H2B/DNA ANAs, it does not develop the full spectrum of ANAs seen in lupus or GN. One could posit that introducing Sle3 onto the B6.NZMc1 strain may quantitatively or qualitatively augment the ANA response, leading to GN. The characterization of B6.NZMc1/c7 bicongenic mice has recently revealed that this is indeed the case (90). 6500 lished observations). With respect to CD22, it is possible that allelic differences between B6 (CD22.2 allele) and NZB/NZW/ NZM2410 strains (CD22.1 allele) may be associated with functional differences in B cell signaling (89). Further work is needed to assess the contributions of these allelic differences to the phenotypes seen in B6.NZMc7 mice and to the pathogenesis of lupus in the NZM2410 strain. Although Sle3 has been described as a single locus in this paper, it is certainly possible that additional genes within this interval are contributing to the observed phenotypes. Ongoing studies with newer congenics bearing shorter genomic intervals are in progress to address the relative contributions of the different regions within the NZM2410-derived chromosome 7 susceptibility interval. Assigning a robust phenotype to these subinterval congenics would greatly boost our efforts to narrow down the vicinity of the culprit gene(s). The eventual identification of the culprit gene(s) on murine chromosome 7 promises to enrich our understanding of how specific defects in T cell function could impact systemic autoimmunity in general and pathogenic ANA formation in particular. Acknowledgments We thank Drs. Eric Sobel and Joel Schiffenbauer for critical review of the manuscript and Feiyan Liu for technical assistance. References 1. Mohan, C., and S. K. Datta. 1995. Lupus: key pathogenic mechanisms and contributing factors. Clin. Immunol. Immunopathol. 77:209. 2. Theofilopoulos, A. N. 1995. The basis of autoimmunity. I. Mechanisms of aberrant self-recognition. Immunol. Today 16:90. 3. Kotzin, B. L. 1996. Systemic lupus erythematosus. Cell 85:303. 4. Wakeland, E. K., L. Morel, C. Mohan, and M. Yui. 1997. Genetic dissection of lupus nephritis in murine models of SLE. J. Clin. Immunol. 17:272. 5. Vyse, T. J., and J. A. Todd. 1996. Genetic analysis of autoimmune disease. Cell 85:311. 6. Kono, D. H., and A. N. Theofilopoulos. 1996. Genetic contributions to SLE. J. Autoimmun. 9:437. 7. Rudofsky, U. H., B. D. Evans, S. L. Balaban, V. D. Mottironi, and A. E. Gabrielsen. 1993. Differences in expression of lupus nephritis in New Zealand Mixed H-2z homozygous inbred strains of mice derived from New Zealand Black and New Zealand White mice: origins and initial characterization. Lab. Invest. 68:419. 8. Mohan, C., L. Morel, P. Yang, and E. K. Wakeland. 1998. Splenic B1a cell expansion in NZM2410 lupus strain, with strong antigen presenting capability. Arthritis Rheum. 41:1652. 9. Morel, L., U. H. Rudofsky, J. A. Longmate, J. Schiffenbauer, and E. K. Wakeland. 1994. Polygenic control of susceptibility to murine systemic lupus erythematosus. Immunity 1:219. 10. Kono, D. H., R. W. Burlingame, D. G. Owens, A. Kuramochi, R. S. Balderas, D. Balomenos, and A. N. Theofilopoulos. 1994. Lupus susceptibility loci in New Zealand mice. Proc. Natl. Acad. Sci. USA 91:10168. 11. Vidal, S., D. H. Kono, and A. N. Theofilopoulos. 1998. Loci predisposing to autoimmunity in MRL-Faslprx and C57BL/6-Faslpr mice. J. Clin. Invest. 101:696. 12. Watson, M. L., J. K. Rao, G. S. Gilkeson, P. Ruiz, E. M. Eicher, D. S. Pisetsky, A. Matsuzawa, J. M. Rochelle, and M. F. Seldin. 1992. Genetic analysis of MRLlpr mice: relationship of the Fas apoptosis gene to disease manifestations and renal disease-modifying loci. J. Exp. Med. 176:1645. 13. Wakeland, E. K., L. Morel, K. Achey, M. E. Yui, and J. A. Longmate. 1998. Speed congenic: a classic technique moves into the fast lane (relatively speaking). Immunol. Today 18:473. 14. Morel, L., Y. Yu, K. R. Blenman, R. A. Caldwell, and E. K. Wakeland. 1996. Production of congenic mouse strains carrying genomic intervals containing SLE-susceptibility genes derived from the SLE-prone NZM2410 strain. Mamm. Genome 7:335. 15. Morel, L., C. Mohan, Y. Yu, B. Croker, X.-H. Tian, A. Deng, and E. K. Wakeland. 1997. Functional dissection of SLE pathogenesis using congenic mouse strains. J. Immunol. 158:6019. 16. Mohan, C., L. Morel, B. Croker, P. Yang, and E. K. Wakeland. 1997. Genetic dissection of SLE pathogenesis: Sle2 on murine chromosome 4 leads to B-cell hyperactivity. J. Immunol. 159:454. 17. Mohan, C., E. Alas, L. Morel, P. Yang, and E. K. Wakeland. 1997. Genetic dissection of SLE pathogenesis: Sle1 on murine chromosome 1 leads to loss of tolerance to H2A/H2B/DNA subnucleosomes. J. Clin. Invest. 101:1362. 18. Mohan, C., S. Adams, V. Stanik, and S. K. Datta. 1993. Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus. J. Exp. Med. 177:1367. 19. Mohan, C., Y. Shi, J. D. Laman, and S. K. Datta. 1995. Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis. J. Immunol. 154:1470. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 differentiation. The importance of these molecules has been elegantly demonstrated by targeted deletion of these molecules in mice. Thus, mutations in Fas/FasL lead to impaired peripheral T cell tolerance and massive lymphoproliferation, with full-blown systemic autoimmunity (on the appropriate genetic background) (55, 56). Knockouts of IL-2, IL-2R, the common cytokine receptor g-chain (gc), and gc–associated tyrosine kinase (Jak3) all result in impaired lymphocytic homeostasis (as exemplified by an age-dependent accumulation of activated/memory CD41, but not CD81, T cells), often accompanied by features of autoimmunity (57– 64). Through the use of TCR transgenic mice, this expansion of activated CD41 T cells in several of these models has been attributed to impaired T cell AICD (65– 69). CTLA4 is yet another molecule that plays a critical role in peripheral T cell tolerance (70), as CTLA42/2 mice display massive lymphoproliferation, overt autoimmunity, and early mortality (71, 72). Additional molecules that impact immune responsiveness, T cell activation, and CD4: CD8 ratios include members of the NF-kB/RelB family, ikB, and ZAP-70 (73–77). Although the above molecules do not map to the study interval on murine chromosome 7, they do suggest that Sle3 could be impinging on the same biochemical pathways underlying T cell expansion and AICD. Signaling studies with B6.NZMc7 mice are currently in progress, to explore this further. Equally important, these other molecules have to be viewed as potential candidate genes for human lupus or for systemic autoimmunity in general. T cells with low anti-self avidity are likely to escape thymic censorship and exit to the periphery, where they are likely to encounter self-Ags. Anti-self T cells with aberrant thresholds for expansion/ cell death (such as those with Sle3 or allelic variants of the other molecules described above), would be more readily triggered into activation and may not be as readily purged from the immune system. Such inefficiency in peripheral T cell tolerance may be expected to manifest differently, depending on the genetic background. Thus, on the B6 background, this might simply lead to a low grade polyclonal T cell activation, leading to serological and cellular phenotypes similar to those observed in B6.NZMc7 mice. On the other hand, the presence of additional susceptibility genes could transform this phenotype into a more pathological one, as discussed above (Fig. 8). Importantly, similar epistatic relationships with background genes have also been described for other molecules that impact T cell homeostasis. For instance, the Faslpr/lpr allele leads to low grade humoral autoimmunity on the B6 background, but to high grade, pathological autoimmunity on the MRL background (39, 55, 78). The study interval on chromosome 7 harbors several candidate genes of immunological interest, including tristetraprolin (TTP), TGF-b, CD22, and Bcl3. TTP2/2 mutant mice exhibit myeloid hyperplasia, cachexia, dermatitis, erosive arthritis, high titers of anti-nuclear (including anti-dsDNA) Abs, and glomerular mesangial thickening (79). CD222/2 mice exhibit increased B cell hyperresponsiveness, elevated serum IgM, serum ANAs, an expansion of B1 cells, and a heightened immune response (80 – 83). Interestingly, TGF-b2/2 mice exhibit multifocal inflammatory disease, activated T cells with elevated CD4:CD8 ratios, a more diffuse splenic architecture, lymphadenopathy, and serum ANAs (84 – 87). On the other hand, aberrant Bcl3 expression has been commonly associated with B cell lymphoproliferation and leukemogenesis (88). As some of the phenotypes impacted by these candidate molecules overlap with those observed in B6.NZMc7 mice, it is important to ascertain whether any of these represent Sle3. Thus far, we have failed to detect any expression differences or coding region polymorphisms in the TTP and TGF-b genes, between B6 and NZM2410/NZW (L. Morel and Y. Yu, unpub- Sle3 IMPACTS T CELL ACTIVATION AND APOPTOSIS The Journal of Immunology 48. Desai-Mehta, A., C. Mao, S. Rajagopalan, T. Robinson, and S. K. Datta. 1995. Structure and specificity of T cell receptors expressed by potentially pathogenic anti-DNA autoantibody-inducing T cells in human lupus. J. Clin. Invest. 95:531. 49. Filaci, G., I. Grasso, P. Contini, M. A. Imro, L. Lanza, M. Scudeletti, E. Rossi, F. Puppo, E. Damasio, and F. Indiveri. 1996. dsDNA-, nucleohistone- and DNase I-reactive T lymphocytes in patients affected by systemic lupus erythematosus: correlation with clinical disease activity. Clin. Exp. Rheumatol. 14:543. 50. Singh, R. R., V. Kumar, F. M. Ebling, S. Southwood, A. Sette, E. E. Sercarz, and B. H. Hahn. 1995. T cell determinants from autoantibodies to DNA can upregulate autoimmunity in murine systemic lupus erythematosus. J. Exp. Med. 181: 2017. 51. Duncan, S. R., R. L. Rubin, R. W. Burlingame, S. B. Sinclair, K. W. Pekny, and A. N. Theofilopoulos. 1996. Intrathymic injection of polynucleosomes delays autoantibody production in BXSB mice. Clin. Immunol. Immunopathol. 79:171. 52. Voll, R. E., E.A. Roth, I. Girkontaite, H. Fehr, M. Herrmann, H. M. Lorenz, and J. R. Kalden. 1997. Histone-specific Th0 and Th1 clones derived from systemic lupus erythematosus patients induce double-stranded DNA antibody production. Arthritis Rheum. 40:2162. 53. Crow, M. K., A. G. DelGiudice, J. B. Zehetbauer, J. L. Lawson, N. Brot, H. Weissbach, and K. B. Elkon. 1994. Autoantigen-specific T cell proliferation induced by the ribosomal P2 protein in patients with systemic lupus erythematosus. J. Clin. Invest. 94:345. 54. Van, P. L., and A. K. Abbas. 1998. Homeostasis and self-tolerance in the immune system: turning lymphocytes off. Science 280:243. 55. Cohen, P. L., and R. A. Eisenberg. 1991. lpr and gld: single gene models of systemic autoimmunity and lymphoproliferative disease. Annu. Rev. Immunol. 9:243. 56. Nagata, S., and T. Suda. 1995. Fas and Fas ligand: lpr and gld mutations. Immunol. Today 16:39. 57. Leonard, W. J., E. W. Shores, and P. E. Love. 1995. Role of the common cytokine receptor Y chain in cytokine signaling and lymphoid development. Immunol. Rev. 148:97. 58. Sadlack, B., J. Lohler, H. Schorle, G. Klebb, H. Haber, E. Sickel, R. J. Noelle, and I. Horak. 1995. Generalized autoimmune disease in interleukin-2-deficient mice is triggered by an uncontrolled activation and proliferation of CD41 T cells. Eur. J. Immunol. 25:3053. 59. Suzuki, H., T. M. Kundig, C. Furlonger, A. Wakeham, E. Timms, T. Matsuyama, R. Schmits, J. J. Simard, P.S. Ohashi, H. Griesser. 1995. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor b. Science 268:1472. 60. DiSanto, J. P., W. Muller, G. D. Guy, A. Fischer, and K. Rajewsky. 1995. Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor g chain. Proc. Natl. Acad. Sci. USA 92:377. 61. Cao, X., E. W. Shores, L. J. Hu, M. R. Anver, B. L. Kelsall, S. M. Russell, J. Drago, M. Noguchi, A. Grinberg, E. T. Bloom, et al. 1995. Defective lymphoid development in mice lacking expression of the common cytokine receptor g chain. Immunity 2:223. 62. Nosaka, T., D. J. van, R. A. Tripp, W. E. Thierfelder, B. A. Witthuhn, A. P. McMickle, P. C. Doherty, G. C. Grosveld, and J. N. Ihle. 1995. Defective lymphoid development in mice lacking Jak3. Science 270:800. 63. Park, S. Y., K. Saijo, T. Takahashi, M. Osawa, H. Arase, N. Hirayama, K. Miyake, H. Nakauchi, T. Shirasawa, and T. Saito. 1995. Developmental defects of lymphoid cells in Jak3 kinase-deficient mice. Immunity 3:771. 64. Nakajima, H., E. W. Shores, M. Noguchi, and W. J. Leonard. 1997. The common cytokine receptor g chain plays an essential role in regulating lymphoid homeostasis. J. Exp. Med. 185:189. 65. Nakajima, H., and W. J. Leonard. 1997. Impaired peripheral deletion of activated T cells in mice lacking the common cytokine receptor g-chain. J. Immunol. 159: 4737. 66. Saijo, K., S. Y. Park, Y. Ishida, H. Arase, and T. Saito. 1997. Crucial role of Jak3 in negative selection of self-reactive T cells. J. Exp. Med. 185:351. 67. DiSanto, J. P., G. D. Guy, A. Fisher, and A. Tarakhovsky. 1996. Critical role for the common cytokine receptor g chain in intrathymic and peripheral T cell selection. J. Exp. Med. 183:1111. 68. Parijs, L. V., A. Biuckians, A. Ibragimov, F. W. Alt, D. M. Willerford, and A. K. Abbas. 1997. Functional responses and apoptosis of CD25 (IL-2Ra)-deficient T cells expressing a transgenic antigen receptor. J. Immunol. 158:3738. 69. Kneitz, B., T. Herrmann, S. Yonehara, and A. Schimpl. 1995. Normal clonal expansion but impaired Fas-mediated cell death and anergy induction in interleukin-2-deficient mice. Eur. J. Immunol. 25:2572. 70. Perez, V. L., L. V. Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, and A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA4 engagement. Immunity 6:411. 71. Tivol, E. A., F. Borriello, A. N. Schweitzer, W. P. Lynch, J. A. Bluestone, and A. H. Sharpe. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3:541. 72. Waterhouse, P., J. M. Penninger, E. Timms, A. Wakeham, A. Shahinian, K. P. Lee, C. B. Thompson, H. Griesser, and T. W. Mak. 1995. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270:985. 73. Esslinger, C. W., A. Wilson, B. Sordat, F. Beermann, and C. V. Jongeneel. 1997. Abnormal T lymphocyte development induced by targeted overexpression of IkB a. J. Immunol. 158:5075. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 20. Nicoletti, I., G. Migliorati, M. C. Pagliacci, F. Grignani, and C. Riccardi. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139:271. 21. Vermes, I., C. Haanen, N. H. Steffens, and C. Reutelingsperger. 1995. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J. Immunol. Methods 184:39. 22. Rozzo, S. J., C. G. Drake, B. L. Chiang, M. E. Gershwin, and B. L. Kotzin. 1994. Evidence for polyclonal T cell activation in murine models of systemic lupus erythematosus. J. Immunol. 153:1340. 23. Jung, L. K., R. A. Good, and G. Fernandes. 1984. In vitro immune response of cells of various lymphoid tissues in (NZB 3 NZW)F1 mice: evidence for abnormality of the mesenteric lymph node cells. J. Immunol. 132:1265. 24. Giese, T., and W. F. Davidson. 1992. Evidence for early-onset, polyclonal activation of T cell subsets in mice homozygous for lpr. J. Immunol. 149:3097. 25. Horwitz, D. A., and W. T. Stohl. 1993. T lymphocytes, cytokines, and immune regulation in systemic lupus erythematosus. In Dubois’ Lupus Erythematosus. D. J. Wallace, and B. H. Hahn, eds. Lea and Febiger, Philadelphia, pp. 83–96. 26. Chen, S. Y., Y. Takeoka, A. A. Ansari, R. Boyd, D. M. Klinman, and M. E. Gershwin. 1996. The natural history of disease expression in CD4 and CD8 gene-deleted New Zealand Black (NZB) mice. J. Immunol. 157:2676. 27. Chesnutt, M. S., B. K. Finck, N. Killeen, M. K. Connolly, H. Goodman, and D. Wofsy. 1998. Enhanced lymphoproliferation and diminished autoimmunity in CD4-deficient MRL/lpr mice. Clin. Immunol. Immunopathol. 87:23. 28. Wofsy, D., and W. E. Seaman. 1985. Successful treatment of autoimmunity in NZB/NZW F1 mice with monoclonal antibody to L3T4. J. Exp. Med. 161:378. 29. Datta, S. K., H. Patel, and D. Berry. 1987. Induction of a cationic shift in IgG anti-DNA autoantibodies: role of T helper cells with classical and novel phenotypes in three murine models of lupus nephritis. J. Exp. Med. 165:1252. 30. Ando, D. G., E. E. Sercarz, and B. H. Hahn. 1987. Mechanisms of T and B cell collaboration in the in vitro production of anti-DNA antibodies in the NZB/NZW F1 murine SLE model. J. Immunol. 138:3185. 31. Sobel, E. S., V. N. Kakkanaiah, M. Kakkanaiah, R. L. Cheek, P. L. Cohen, and R. A. Eisenberg. 1994. T-B collaboration for autoantibody production in lpr mice is cognate and MHC-restricted. J. Immunol. 152:6011. 32. Shivakumar, S., G. C. Tsokos, and S. K. Datta. 1989. T cell receptor a/b expressing double-negative (CD42/CD82) and CD41 T helper cells in humans augment the production of pathogenic anti-DNA autoantibodies associated with lupus nephritis. J. Immunol. 143:103. 33. Vassilopoulos, D., B. Kovacs, and G. C. Tsokos. 1995. TCR/CD3 complexmediated signal transduction pathway in T cells and T cell lines from patients with systemic lupus erythematosus. J. Immunol. 155:2269. 34. Liossis, S. N., X. Z. Ding, G. J. Dennis, and G. C. Tsokos. 1998. Altered pattern of TCR/CD3-mediated protein-tyrosyl phosphorylation in T cells from patients with systemic lupus erythematosus: deficient expression of the T cell receptor z chain. J. Clin. Invest. 101:1448. 35. Tsokos, G. C. 1992. Lymphocyte abnormalities in human lupus. Clin. Immunol. Immunopathol. 63:7. 36. Dayal, A. K., and G. M. Kammer. 1996. The T cell enigma in lupus. Arthritis Rheum. 39:23. 37. Kammer, G. M., I. U. Khan, J. A. Kammer, I. Olorenshaw, and D. Mathis. 1996. Deficient type I protein kinase A isozyme activity in systemic lupus erythematosus T lymphocytes. II. Abnormal isozyme kinetics. J. Immunol. 157:2690. 38. Bossu, P., G. G. Singer, P. Andres, R. Ettinger, A. Marshak Rothstein, and A. K. Abbas. 1993. Mature CD41 T lymphocytes from MRL/lpr mice are resistant to receptor-mediated tolerance and apoptosis. J. Immunol. 151:7233. 39. Izui, S., V. E. Kelley, K. Masuda, H. Yoshida, J. B. Roths, and E. D. Murphy. 1984. Induction of various autoantibodies by mutant gene lpr in several strains of mice. J. Immunol. 133:227. 40. Kovacs, B., D. Vassilopoulos, S. A. Vogelgesang, and G. C. Tsokos. 1996. Defective CD3-mediated cell death in activated T cells from patients with systemic lupus erythematosus. Clin. Immunol. Immunopathol. 81:293. 41. Budagyan, V. M., E. G. Bulanova, N. I. Sharova, M. F. Nikonova, M. L. Stanislav, and A. A. Yarylin. 1998. The resistance of activated T-cells from SLE patients to apoptosis induced by human thymic stromal cells. Immunol. Lett. 60:1. 42. Sabzevari, H., S. Propp, D. H. Kono, and A. N. Theofilopoulos. 1997. G1 arrest and high expression of cyclin kinase and apoptosis inhibitors in accumulated activated/memory phenotype CD41 cells of older lupus mice. Eur. J. Immunol. 27:1901. 43. Elkon, K. B. 1994. Apoptosis in SLE: too little or too much? Clin. Exp. Rheumatol. 12:553. 44. Kolowos, W., M. Herrmann, B. B. Ponner, R. Voll, P. Kern, C. Frank, and J. R. Kalden. 1997. Detection of restricted junctional diversity of peripheral T cells in SLE patients by spectratyping. Lupus 6:701. 45. Mato, T., K. Masuko, Y. Misaki, N. Hirose, K. Ito, Y. Takemoto, K. Izawa, S. Yamamori, T. Kato, K. Nishioka, and K. Yamamoto. 1997. Correlation of clonal T cell expansion with disease activity in systemic lupus erythematosus. Int. Immunol. 9:547. 46. Holbrook, M. R., P. J. Tighe, and R. J. Powell. 1996. Restrictions of T cell receptor b chain repertoire in the peripheral blood of patients with systemic lupus erythematosus. Ann. Rheum. Dis. 55:627. 47. Kaliyaperumal, A., C. Mohan, W. Wu, and S. K. Datta. 1996. Nucleosomal peptide epitopes for nephritis-inducing T helper cells of murine lupus. J. Exp. Med. 183:2459. 6501 6502 83. Otipoby, K. L., K. B. Andersson, K. E. Draves, S. J. Klaus, A. G. Farr, J. D. Kerner, R. M. Perlmutter, C. L. Law, and E. A. Clark. 1996. CD22 regulates thymus-independent responses and the lifespan of B cells. Nature 384:634. 84. Christ, M., F. N. McCartney, A. B. Kulkarni, J. M. Ward, D. E. Mizel, C. L. Mackall, R. E. Gress, K. L. Hines, H. Tian, S. Karlsson. 1994. Immune dysregulation in TGF-b1-deficient mice. J. Immunol. 153:1936. 85. Kulkarni, A. B., C. G. Huh, D. Becker, A. Geiser, M. Lyght, K. C. Flanders, A. B. Roberts, M. B. Sporn, J. M. Ward, and S. Karlsson. 1993. Transforming growth factor b1 null mutation in mice causes excessive inflammatory response and early death. Proc. Natl. Acad. Sci. USA 90:770. 86. Dang, H., A. G. Geiser, J. J. Letterio, T. Nakabayashi, L. Kong, G. Fernandes, and N. Talal. 1995. SLE-like autoantibodies and Sjögren’s syndrome-like lymphoproliferation in TGF-b knockout mice. J. Immunol. 155:3205. 87. Shull, M. M., I. Ormsby, A. B. Kier, S. Pawlowski, R. J. Diebold, M. Yin, R. Allen, C. Sidman, G. Proetzel, D. Calvin. 1992. Targeted disruption of the mouse transforming growth factor-b1 gene results in multifocal inflammatory disease. Nature 359:693. 88. Ong, S. T., M. L. Hackbarth, L. C. Degenstein, D. A. Baunoch, J. Anastasi, and T. W. McKeithan. 1998. Lymphadenopathy, splenomegaly, and altered immunoglobulin production in BCL3 transgenic mice. Oncogene 16:2333. 89. Nadler, M. J., P. A. McLean, B. G. Neel, and H. H. Wortis. 1997. B cell antigen receptor-evoked calcium influx is enhanced in CD22-deficient B cell lines. J. Immunol. 159:4233. 90. Mokan, C., L. Morel, P. Yang, H. Watanabe, B. Croker, G. Gilkeson, and E. K. Wakeland. 1999. Genetic dissection of lupus pathogenesis: a recipe for nephrophilic autoantibodies. J. Clin. Invest. In press. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 74. Boothby, M. R., A. L. Mora, D. C. Scherer, J. A. Brockman, and D. W. Ballard. 1997. Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-kB. J. Exp. Med. 185:1897. 75. Weih, F., G. Warr, H. Yang, and R. Bravo. 1997. Multifocal defects in immune responses in RelB-deficient mice. J. Immunol. 158:5211. 76. Chan, A. C., T. A. Kadlecek, M. E. Elder, A. H. Filipovich, W. L. Kuo, M. Iwashima, T. G. Parslow, and A. Weiss. 1994. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science 264: 1599. 77. Elder, M. E., D. Lin, J. Clever, A. C. Chan, T. J. Hope, A. Weiss, and T. G. Parslow. 1994. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science 264:1596. 78. Hang, L. M., M. T. Aguado, F. J. Dixon, and A. N. Theofilopoulos. 1985. Induction of severe autoimmune disease in normal mice by simultaneous action of multiple immunostimulators. J. Exp. Med. 161:423. 79. Taylor, G. A., E. Carballo, D. M. Lee, W. S. Lai, M. J. Thompson, D. D. Patel, D. I. Schenkman, G. S. Gilkeson, H. E. Broxmeyer, B. F. Haynes, and P. J. Blackshear. 1996. A pathogenetic role for TNFa in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity 4:445. 80. O’Keefe, T. L., G. T. Williams, S. L. Davies, and M. S. Neuberger. 1996. Hyperresponsive B cells in CD22-deficient mice. Science 274:798. 81. Sato, S., A. S. Miller, M. Inaoki, C. B. Bock, P. J. Jansen, M. L. Tang, and T. F. Tedder. 1996. CD22 is both a positive and negative regulator of B lymphocyte antigen receptor signal transduction: altered signaling in CD22-deficient mice. Immunity 5:551. 82. Nitschke, L., R. Carsetti, B. Ocker, G. Kohler, and M. C. Lamers. 1997. CD22 is a negative regulator of B-cell receptor signalling. Curr. Biol. 7:133. Sle3 IMPACTS T CELL ACTIVATION AND APOPTOSIS