Alterations in splenic architecture and the localization of anti-double-stranded DNA B cells in aged mice

Ashlyn S. Eaton-Bassiri, Laura Mandik-Nayak, Su-jean Seo, Michael P. Madaio1, Michael P. Cancro2 and Jan Erikson

The Wistar Institute, Philadelphia, PA 19104, USA
1 Departments of 1Medicine, and
2 Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Correspondence to: J. Erikson


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Aging is characterized by a decline in humoral immunity and a concommitant increased incidence of anti-DNA and other autoantibodies. To define how the regulation of autoreactive B cells is altered with age, we have used BALB/c mice with an Ig heavy H chain transgene to track the fate of anti-double-stranded (ds) DNA B cells in vivo. In young adult mice, anti-dsDNA B cells are developmentally arrested and excluded from the splenic B cell follicle, whereas in most aged mice they are mature and localize within the B cell follicle. Furthermore, we have detailed global changes in lymphoid architecture that accompany aging: CD4+ T cells are found not only in the periarteriolar lymphoid sheath, but also in the B cell follicles. Strikingly, these disruptions are similar to those that precede serum anti-dsDNA antibody expression in autoimmune MRL-lpr/lpr mice.

Keywords: anti-nuclear antibodies, autoimmunity, nephritis, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Aging in humans and mice has been associated with a decline in the humoral immune response to foreign antigens (14). The decline in protective immunity is manifested by an increased susceptibility to disease and a lowered response to vaccination (58). At the same time, an increased incidence of anti-nuclear antibodies (ANA), including anti-DNA, and other autoantibodies has also been documented (912).

The etiology of these dramatic changes in immune responsiveness is poorly understood. Furthermore, little is known about how age affects the ability to maintain both B and T cell tolerance to self antigens. Adoptive transfer studies in which SCID mice were reconstituted with lymphocytes from young or old mice prior to immunization showed that both aged T and B cells can influence the quality of the B cell immune response (13). Several groups have reported changes in the phenotype of T cells upon aging (1419). In both humans and mice, the proportion of T cells expressing a naive phenotype decreases, while the proportion exhibiting an activated/memory phenotype increases with age. Although Fas expression is known to be elevated on activated T cells (20), its expression pattern and function on T cells from aged mice remain controversial. One group reported a decline in Fas levels and function in aged mice (21), but this observation has been refuted by others (2224). Functionally, both memory and naive T cells from aged animals, relative to young controls, have been shown to be hyporesponsive to a number of stimuli (14,19,25,26).

Fewer phenotypic and functional changes have been described for B cells from aged mice. Analyses of murine bone marrow (BM) have demonstrated an age-associated decline in B lymphopoiesis, particularly in the pre-B cell compartment (2729). Kline et al. also found a decrease in the number of immature B cells in the BM and spleens of aged mice. However, despite the decrease in developing B cells, the relative number of mature peripheral B cells was not dramatically decreased (29,30). In vivo labeling studies suggest that the lifespan of peripheral B cells in aged mice is extended (2,30). Whether alterations in B cell progenitor pool sizes are related to lifespan changes of peripheral B cells is not known. It has been hypothesized that changes in the development/maturation of B cells may in turn affect the ability to maintain B cell tolerance to self-antigens (29).

Our laboratory has developed an approach to follow the fate of anti-double-stranded (ds) DNA B cells in the context of a diverse repertoire (31). To directly determine how age affects the regulation of autoreactive B cells, we have utilized BALB/c mice with the VH3H9 H chain transgene (Tg). The VH3H9 Tg not only allows the analysis of endpoints such as serum autoantibodies, but also allows tracking of the anti-dsDNA B cells themselves in vivo. In this study, we find several points at which the regulation of anti-dsDNA B cells differs between old and young mice.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Aged C57BL/6, DBA/2 and BALB/c mice were obtained from either the National Institute of Aging colony maintained by Charles River (Kingston, NY) or were purchased as young animals from the Jackson Laboratory (Bar Harbor, ME) (C57BL/6 and DBA/2) and Harlan Sprague Dawley (Indianapolis, IN) (BALB/c), and allowed to age in an isolated specific pathogen-free room at The Wistar Institute animal facility. Young control mice were housed under the same conditions. One cohort of aged C57BL/6 mice (13–14 months) and young controls (2 months) were obtained from an isolated specific pathogen-free animal colony at the University of Pennsylvania. No difference was detected between mice obtained from the different colonies. The VH3H9 Tg, described previously (32), was backcrossed onto the BALB/c background for at least 15 generations, and mice were bred and maintained along with Tg controls in the animal facility at The Wistar Institute. The presence of the VH3H9 Tg was confirmed by PCR amplification of tail DNA with primers specific for VH3H9 (32).

ANA assay
ANA were detected in serum samples as previously described (33). Briefly, homogenous nuclear (HN) ANA binding to HEP-2 cells (Antibodies Inc., Davis, CA) was detected with a combination of FITC-conjugated goat anti-mouse IgG plus anti-mouse IgM reagents (Southern Biotechnology, Birmingham, AL). For H chain isotyping of ANA, class-specific reagents were used individually. The presence of Ig{lambda} ANA was determined on serum samples diluted 1:10 using goat anti-mouse Ig{lambda}–FITC (Southern Biotechnology) as previously described (31). ANA titer equaled the reciprocal of the last dilution still presenting an HN staining pattern. All samples were tested at an initial dilution of 1:100 and serially diluted. Serum that was ANA at 1:100 remained ANA when tested at a dilution of 1:10.

Crithidia luciliae assay
The presence of anti-dsDNA antibodies in serum samples was detected using fixed-permeabilized C. luciliae protozoans as the substrate (Antibodies Inc.). In this assay, anti-dsDNA antibodies are identified by their ability to bind the kinetoplast (34). Serum samples were tested at a 1:100 dilution followed by FITC-conjugated goat anti-mouse IgG plus anti-mouse IgM reagents (Southern Biotechnology).

Histological evaluation of nephritis
Kidneys from VH3H9 and Tg BALB/c, C57BL/6, DBA/2 and MRL-lpr/lpr mice were fixed in 10% buffered formalin (Fisher Scientific, Pittsburgh, PA), and embedded in paraffin. Sections (4 µm) of each sample were stained with hematoxylin & eosin (H & E). Kidney sections were assessed using light microscopy by one of the authors (M. P. M.) without prior knowledge of mouse genotype or age. Disease severity was graded according to previously described methods (35), where glomerulonephritis, interstitial nephritis and vasculitis were scored separately.

Immunohistochemistry
Spleen sections were prepared and stained according to the protocol described in Jacob et al. (36). Briefly, sections were blocked using PBS/5% normal goat serum (Sigma, St Louis, MO)/0.1% Tween 20 (Sigma), and then stained with Cy34.1–FITC (anti-CD22) (PharMingen, San Diego, CA) and either GK1.5–biotin (anti-CD4) or 53-6.72–biotin (anti-CD8) (ATCC, Manassas, VA). Anti-FITC– horseradish peroxidase (HRP) (Chemicon, Temecula, CA) and streptavidin–alkaline phosphatase (AP) (Southern Biotechnology) were used as secondary reagents. HRP and AP were developed using the substrates 3-amino-9-ethyl-carbazole (Sigma) and Fast-Blue BB base (Sigma) respectively. For Ig{lambda} staining, either pan anti-{lambda}–AP (Southern Biotechnology) or LS136–biotin (anti-{lambda}1; gift of Garnett Kelsoe, Duke University, Durham, NC)/streptavidin–AP was used in conjunction with Cy34.1–FITC/anti-FITC–HRP. For architectural grading, three or more follicles on greater than three sections from each mouse were graded in blind fashion by two separate readers. Syndecan-1 staining was performed as previously described (33) using 281-2 (anti-syndecan-1) (PharMingen), MAR18.5–biotin (anti-rat Ig, grown as supernatant) and streptavidin–AP.

Flow cytometry analysis
Spleens were removed from aged and young mice, single-cell suspensions prepared, and erythrocytes removed by hypotonic lysis. Cells (1x106) were surface stained according to standard protocols (37). The following antibodies/reagents were used: RA3-6B2–phycoerythrin (PE) or –biotin (anti-B220), 2C11–FITC (anti-CD3), Jo2–PE (anti-Fas), 7G6–FITC (anti-CD21/35), Cy34.1–FITC (anti-CD22), B3B4–FITC (anti-CD23), IM7–FITC (anti-CD44), Mel-14–FITC or –PE (anti-CD62L), R11-153–FITC (anti-{lambda}1), R26-46–FITC (anti-{lambda}1 + {lambda}2), M1/69–FITC (anti-HSA) and 2G9–FITC (anti-I-Ad/I-Ed) all purchased from PharMingen; JC5.1–PE (anti-{lambda}; gift of J. Kearney, University of Alabama, Birmingham, AL); polyclonal anti-IgM–PE (Southern Biotechnology); streptavidin–Red670 (Gibco/BRL, Gaithersburg, MD). All samples were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software. Based on live lymphocyte gates from forward and side scatter, 15,000–40,000 events were collected for each sample.

Identification of VH3H9/V{lambda}1 (anti-dsDNA) B cells
The VH3H9 H chain Tg has been shown to be a good excluder of endogenous H chain rearrangement in young adult BALB/c mice (32). Given that allelic exclusion of some Ig Tg is lost as mice age (38), we documented that exclusion was maintained in aged VH3H9 BALB/c mice. All B cells in aged VH3H9 BALB/c mice, including the VH3H9/V{lambda}1 B cells in particular, were IgD by flow cytometry and immunohistochemistry consistent with their exclusive use of the IgM H chain only VH3H9 Tg (data not shown). Several different reagents were used to track {lambda}+ and {lambda}1 B cells (LS136, JC5, R26-46 and R11-153). Using these reagents and flow cytometry, we have shown that >80% of the {lambda}+ B cells in young adult VH3H9 BALB/c mice are {lambda}1 (31). This was also true in aged VH3H9 BALB/c mice (data not shown). Therefore, in most of the analysis, VH3H9/V{lambda}1 anti-dsDNA B cells were followed using pan anti-{lambda} reagents.

Statistical analysis
Statistical significance was determined using either the unpaired Student's t-test or non-parametric Mann–Whitney test with Microsoft Excel and Instat software.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Autoantibody production in aged mice
To better characterize the development of autoantibodies in aging mice, we have determined the incidence of ANA and anti-dsDNA antibodies in several strains. These studies extend previous observations on the production of ANA in aged mice by demonstrating a strain dependence to this process (10,39,40). Regardless of age, ANA were not detected in the serum of DBA/2 mice. ANA were present in the serum of between 40 and 60% of C57BL/6 and BALB/c mice >=17 months of age (Fig. 1A and BGo). The HN staining pattern of these ANA is indistinguishable from that seen in most systemic lupus erythematosus (SLE) patients and in the MRL-lpr/lpr mouse, a murine model of SLE. This staining pattern has been correlated with the presence of anti-dsDNA, anti-histone and anti-chromatin autoantibodies (41). Similar to those in MRL-lpr/lpr mice, ANA in aged BALB/c and C57BL/6 mice were of the IgG subclass (data not shown) (42). The frequency of ANA in aged mice was well below the 100% incidence seen in diseased MRL-lpr/lpr mice. Furthermore, ANA titers were significantly lower in aged C57BL/6 (P <= 0.0001) and BALB/c (P = 0.0002) mice relative to MRL-lpr/lpr mice (Fig. 2Go). Using the C. luciliae assay to distinguish anti-dsDNA antibodies (Fig. 1CGo), all ANA+ MRL-lpr/lpr mice, by age 16–20 weeks, contain anti-dsDNA antibodies in their sera. Anti-dsDNA antibodies were also detectable in sera from aged non-autoimmune C57BL/6 and BALB/c mice, albeit at a lower incidence than in MRL-lpr/lpr mice (Fig. 1DGo).



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Fig. 1 ANA and anti-dsDNA antibody production in aged mice. (A) Representative HN+ ANA+ (top) and ANA (bottom) staining patterns from serum of aged non-autoimmune and autoimmune-prone MRL-lpr/lpr mice. Staining of both the nucleus of non-dividing HEP-2 cells as well as mitotic figures can be seen in the ANA+ picture. (B) Aged mice produce HN+ ANA, which begin to be detectable by 9–12 months. The percentage of animals expressing HN+ ANA was graphed according to age. ANA were developed with anti-IgM–FITC plus anti-IgG–FITC. C57BL/6 ({circ}), VH3H9 BALB/c ({blacksquare}), Tg BALB/c ({square}) and DBA/2 ({blacktriangleup}) mice are presented in conjunction with MRL-lpr/lpr mice (.). The number of animals analyzed at each time point is indicated. n >=10 for all animals measured at 1 month. (C) Representative Crithidia staining of aged non-autoimmune and MRL-lpr/lpr serum. Staining of the kinetoplast (small circular body toward the tail of the animal, denoted by the arrows) indicates the presence of anti-dsDNA antibodies (top), compared to a negative control (bottom). (D) Aged mice express anti-dsDNA antibodies. Sera (from B) were tested for anti-dsDNA antibodies in the C. luciliae assay, and the results graphed according to age and genotype. Anti-IgM–FITC plus anti-IgG–FITC were used to visualize the autoantibodies. C57BL/6 ({circ}), VH3H9 BALB/c ({blacksquare}) and Tg BALB/c ({square}) mice are presented in conjunction with MRL-lpr/lpr mice (.). The number of sera tested at each timepoint is listed in (B).

 


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Fig. 2. Low ANA titers in aged mice. Sera from ANA+ aged non-autoimmune (13–24 months) and MRL-lpr/lpr (3–6 months) mice were titered. ANA titer was defined as the reciprocal of the last dilution still giving an HN+ ANA staining pattern. ANA were developed with anti-IgM–FITC plus anti-IgG–FITC. Each point represents one animal and group averages are indicated by the stars. ANA titers in MRL-lpr/lpr mice (geometric mean titer = 8913) were significantly greater than those titers in aged C57BL/6 (geometric mean titer = 251) (P < 0.0001), VH3H9 (geometric mean titer = 229) (P < 0.0001) and Tg BALB/c (geometric mean titer = 562) (P = 0.0002) mice.

 
To evaluate changes in anti-dsDNA B cells that occur with age, we have used VH3H9 Ig Tg BALB/c mice. The VH3H9 H chain was isolated from anti-DNA antibodies of diseased MRL-lpr/lpr mice (43), and has been shown to pair with endogenous light L chains to create both ANA+ and non-ANA specificities (44,45). The VH3H9 Tg paired with the V{lambda}1 L chain generates an anti-dsDNA antibody with an HN ANA staining pattern (31,44). Using Ig{lambda}-specific reagents, we can follow the fate of anti-dsDNA B cells in vivo in the context of the rest of the B cells in aging VH3H9 mice (31,33). Thus, the VH3H9/V{lambda}1 B cells can be studied as a prototype for ANA B cells in aged mice.

ANA are not present in the serum of young adult VH3H9 mice (32 and Fig. 1BGo); however, as the mice age, serum ANA begin to appear (Fig. 1BGo). In contrast to the IgG ANA found in aged Tg BALB/c and C57BL/6 mice, ANA in aged VH3H9 mice are exclusively IgM (data not shown), presumably due to the constraints of the IgM-only Tg. With that exception, ANA in aged VH3H9 mice are similar in incidence (Fig. 1BGo) and titer (Fig. 2Go) to those in age-matched Tg BALB/c mice. Furthermore, the proportion of animals expressing anti-dsDNA antibodies was not different, thus suggesting that the presence of the VH3H9 Tg did not alter the onset or quality of ANA produced. Given these results, the VH3H9 transgenic mice are a good model for following the fate of ANA B cells in aging.

ANA and nephritis in aged mice
Several studies have described nephritis in aged rats and mice (4648); however, whether this nephritis correlated with autoantibody titers was not determined. We have evaluated the degree of nephritis in kidneys from young and aged ANA+ and ANA mice (Table 1Go). Histological analysis shows that young mice display limited nephritis by these criteria. Aged VH3H9 mice display a wide range of nephritis which, on average, is not significantly different from that of young VH3H9 mice (P = 0.51) (Table 1Go). However, aged C57BL/6, DBA/2 and Tg BALB/c mice (17–24 months) develop significant nephritis relative to their younger (2–8 months) counterparts (P = 0.001, P = 0.0008 and P = 0.0007 respectively). The restricted nature of the ANA produced by the aged VH3H9 BALB/c mice compared to Tg BALB/c mice may be responsible for this difference. Overall, the pathology detected in aged mice was less severe than that present in 4–6.5 month MRL-lpr/lpr mice (P <= 0.0007) (Table 1Go). Nephritis has been correlated with ANA titers in MRL-lpr/lpr mice (reviewed in 49). Clearly, however, ANA are not required for the age-associated nephritis described here, as aged DBA/2 mice develop significant nephritis without the production of detectable ANA (Fig. 1BGo and Table 1Go).


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Table 1. Development of nephritis with age
 
Changes in localization and surface phenotype of anti-dsDNA B cells in aged mice
Taking advantage of the VH3H9 Tg to follow anti-dsDNA B cells, we have previously correlated the absence of anti-dsDNA serum antibodies in young adult VH3H9 BALB/c mice with the follicular exclusion and developmental arrest of anti-dsDNA B cells (31). When anti-dsDNA B cells were examined in aged VH3H9 mice, striking differences in their localization were noted in mice aged >=11 months (Fig. 3Go). In seven (60%) of 12 aged VH3H9 mice, the anti-dsDNA B cells no longer localize to the T–B junction, but rather they populate the B cell follicle.



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Fig. 3. Localization of the VH3H9/V{lambda}1 anti-dsDNA B cells is altered in aged VH3H9 BALB/c animals. Spleen sections from VH3H9 mice were stained with anti-CD22–FITC/anti-FITC–HRP (orange) and anti-{lambda}–AP (blue). Representatives of the two different splenic localization patterns of anti-dsDNA B cells are shown in (A) and (B). In young adult animals (2–8 months) and 40% of aged animals (>=11 months), the {lambda}1+ anti-dsDNA B cells (blue) localize to the T–B interface of the splenic white pulp (A). In contrast, in 60% of aged VH3H9 animals, the {lambda}1+ anti-dsDNA B cells localize within the B cell follicle (orange area) (B). These are representative sections of a minimum n = 12 for each age range.

 
To determine if the altered localization of anti-dsDNA B cells in aged VH3H9 mice coincided with a change in their surface phenotype, the expression of a panel of B cell developmental and activation markers (Ig, MHC class II, CD21/35, CD22, CD23, CD44, CD62L, B220) was assessed. In all age ranges examined, Ig{lambda}+ B cells were present in the periphery with a decreased level of surface Ig and CD21/35 (Fig. 4A and BGo) (31). We and others have interpreted a low level of Ig and CD21/35 to be an indication of previous encounter with antigen (31,5057). Curiously, anti-dsDNA B cell localization correlated with the surface density of CD21/35 on the surrounding {kappa}-bearing B cells: when anti-dsDNA (Ig{lambda}+) B cells were at the T–B interface, CD21/35 density on the Ig{kappa}+ B cells from that mouse was like that of mature B cells; however, when anti-dsDNA B cells were in the follicle, CD21/35 levels on the surrounding Ig{kappa}+ population were low (down 2.6 ± 0.6-fold) (n = 7).



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Fig. 4. Surface phenotype of Ig{lambda}+ B cells from old and young VH3H9 and Tg mice. Spleen cells from young VH3H9 and Tg BALB/c (2–8 months), and aged VH3H9 BALB/c mice (>=11 months), where anti-dsDNA B cells were localized at either the T–B interface (n = 5) or in the B cell follicle (n = 7), were compared for CD21/35, CD22 and B220 expression levels. Representative dot-plots (A) show relative B220 and Ig{lambda} expression levels on the various mice; note decreased level of Ig{lambda} on anti-dsDNA B cells from VH3H9 mice. The mean fluorescence intensity (MFI) for {lambda} expression is indicated. Representative histograms (B) show CD21/35, CD22 and B220 staining of the total B cell population (gated on B220+ cells, thin line) and Ig{lambda}+ B cells (bold line). No difference was detected for the panel of surface markers on Ig{lambda}+ B cells from young and aged Tg BALB/c mice (data not shown); therefore, representative results from a young mouse are shown. MFI for both the total B cell population (non-bold numbers) and the Ig{lambda}+ B cells (bold numbers) are shown.

 
In aged VH3H9 BALB/c mice, the anti-dsDNA B cells themselves showed marked differences in B220 and CD22 expression that correlated with their splenic localization (Fig. 4BGo). When anti-dsDNA B cells were at the T–B interface, the Ig{lambda}+ anti-dsDNA B cells expressed low levels of B220 (down 2 ± 0.46-fold; P = 0.008) and CD22 (down 2 ± 0.14-fold; P = 0.008) relative to the non-{lambda} B cells in the mouse. This cell surface phenotype is like the immature B cell phenotype documented for young adult VH3H9 BALB/c mice where the anti-dsDNA B cells are also at the T–B interface (Fig. 4BGo) (31). By contrast, when anti-dsDNA B cells were localized in the B cell follicle, they expressed levels of B220 (down 0.13 ± 0.1-fold; P = 0.98) and CD22 (down 0.1 ± 0.05-fold; P = 0.76) comparable to that of the surrounding mature B cells.

To determine if anti-dsDNA B cells were forming antibody-secreting cells (ASC) in aged VH3H9 mice, spleen sections were examined for the presence of Ig{lambda}+ ASC, and serum antibody was analyzed for the L chain isotype of ANA. The threshold of detection of these two assays is likely different: while immunohistochemistry can identify a single plasma cell, many may be required for anti-dsDNA antibodies to be detected in the serum. Unlike in young adult VH3H9 mice, nine of 12 aged VH3H9 mice (>=11 months) had large dark staining Ig{lambda}+ cells in the red pulp suggestive of anti-dsDNA ASC (Fig. 3BGo). In eight of these nine mice, staining of the dark Ig{lambda}+ cells co-localized with syndecan-1 staining, a cell surface proteoglycan expressed on B cells that have differentiated into ASC (58) (data not shown). Furthermore, when sera from these mice were tested by immunofluorescence for the L chain isotype of ANA, two of the nine mice with Ig{lambda}+ ASC in the spleen had Ig{lambda}+ ANA. In these two mice, the anti-dsDNA B cells expressed mature levels of B220 and CD22, and localized in the B cell follicle. These data suggest that while a relocation of the autoreactive Ig{lambda}+ B cells into the follicle and a change in their maturation state are not sufficient to promote ASC formation, they may be necessary.

Aged mice develop disruptions in splenic architecture
To determine if altered splenic localization was unique to the anti-dsDNA B cell population or whether overall splenic architecture also changes with age, spleen sections from young and old mice were stained with anti-CD22 and either anti-CD4 or -CD8 antibodies. Examples of the range of disruption observed in aged mice are shown in Fig. 5Go. In young mice, CD4+ and CD8+ T cells remain tightly compacted around the central arteriole in the periarteriolar lymphoid sheath (PALS) having limited overlap with the B cell follicular area, here defined as grade 0 (Fig. 5Go). In the majority of aged animals analyzed, CD8+ T cells continued to exhibit this restricted localization (grades 0–2) (Fig. 5Go). In contrast, CD4+ T cells in many aged mice were found not only in the PALS but also in the B cell follicle (grades 1–3) (Fig. 5Go). In the spleens of animals with severely disrupted architecture (grade 3), CD8 localization was also altered.



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Fig. 5. Splenic architecture is disrupted in aged animals. Spleen sections from adult (2–8 months) and aged (>9 months) mice were stained with anti-CD22–FITC/anti-FITC–HRP (orange) and with either anti-CD4 or -CD8–biotin/streptavidin–AP (blue). CD22 marks the B cell area, and CD4 and CD8 define the PALS area. In many aged animals, CD4 staining overlaps with the CD22 staining indicating that T cells are present in the B cell follicle. Representative serial sections of each grade are shown. A minimum of five mice from each age range were tested and the cumulative results are shown in Fig. 6Go. Mice were graded using the following scale: 0 = CD4+ and CD8+ T cells remain tightly compacted around the central arteriole; 1 = limited number of CD4+ T cells localized in B area with CD8+ T cells in the PALS; 2 = CD4+ T cells greatly overlap with B cells in follicle, but CD8+ T cells still in PALS; 3 = CD4+ and CD8+ T cells populate both the PALS and the B cell follicle.

 
Results for several strains of mice spanning three age ranges are summarized in Fig. 6Go. Aged DBA/2 mice which are ANA showed no evidence of such alterations (Fig. 6Go). Furthermore, only 36% of Tg BALB/c mice had disrupted CD4+ T cell localization compared to 92% of C57BL/6 mice (9–24 months). Thus, the degree of architectural disruption was influenced by the background strain of the mouse. It is unclear why DBA/2 mice are resistant to age-associated ANA production and changes in splenic architecture. This disparity between strains is interesting, however, in light of the documented resistance of DBA/2 mice to induction of experimental allergic orchitis (59).



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Fig. 6. A spectrum of architecture disruption in aged VH3H9 BALB/c, Tg BALB/c, C57BL/6 and DBA/2 mice. Staining was graded using the scale depicted in Fig. 5Go, and then plotted according to age and genotype. Each symbol represents one mouse and group averages are indicated by the stars. Filled circles indicate mice that were ANA+ at a 1:100 serum dilution; open circles were ANA mice. Both VH3H9 BALB/c (P = 0.0001) and C57BL/6 (P = 0.001) mice develop significant architectural disruptions with age. Aged DBA/2 mice (17–24 months) do not develop disrupted architecture.

 
Clearly, changes in splenic architecture were not required for ANA production as several ANA+ aged animals had no detectable architectural disruption (Fig. 6Go). The presence of the VH3H9 Tg accelerated the changes in CD4+ T cell localization as evidenced by the increased proportion of VH3H9 BALB/c mice with disruptions (93%) over Tg BALB/c (36%). Although CD4+ T cells can be found in the follicle when participating in a germinal center (GC) response (60,61), it is unlikely that most of the infiltrating CD4+ T cells are GC T cells for two reasons: (i) they are not localized in discrete clusters as would be the case for T cells associated with a GC (Fig. 5Go) and (ii) PNA staining of serial sections did not reveal co-localizing GC B cells (data not shown).

Disrupted architecture does not correlate with CD4+ T cell activation
Several groups have reported changes in T cell phenotype, where the proportion of naive T cells decreases and the proportion of activated/memory T cells increases with age (1419). Given that activated T cells participating in an immune response have been shown to localize in the B cell follicle (62,63), the accumulation of T cells expressing an activated/memory phenotype in aged mice could contribute to the changes in cellular localization seen here. We found that this was not the case. C57BL/6 but not DBA/2 mice have age-associated changes in splenic architecture. However, analysis of T cells from aged C57BL/6 and DBA/2 mice confirmed naive to activated/memory phenotype shifts in CD44 and CD62L expression for both strains (Fig. 7AGo and Table 2Go). Likewise, similar shifts in CD62L expression were present in all aged Tg BALB/c mice, even those with no detectable splenic architectural changes (data not shown). Therefore, these data argue against the possibility that significant splenic architectural disruptions in aged mice are solely the result of the increased proportion of T cells expressing an activated/memory phenotype.




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Fig. 7. (A) Acquisition of activated/memory CD4+ T cell phenotype with age. Representative CD62L/CD44 dot plots from CD4+ T cell lymphocyte gates of 2 and 22 month DBA/2 mice. Data from this analysis was used to generate Table 2Go where naive cells were CD62Lhi/CD44lo (upper left quadrant), activated cells were CD62Lhi/CD44hi (upper right quadrant) and memory cells were CD62Llo/CD44hi (lower right quadrant). The results presented are representative of all strains of mice examined where n = 8 for each genotype. (B) T and B cell Fas expression does not decrease with age. Spleen cells from young (2.5 months) and aged (9–25 months) mice were stained with anti-CD3–FITC, anti-Fas–PE and anti-B220–biotin/streptavidin–Red670. The results presented are representative of all strains of mice examined. The histograms on the bottom were generated from the dot plots above, where gates are drawn for B220+ (R2) or CD3+ (R3) cells within the lymphoid gate. The histogram shows Fas expression on lymphocytes from young (thin line) and aged (bold line) animals. The MFI for the T cell histogram is indicated. These are representative plots with a minimum of n = 10 for each genotype.

 

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Table 2. C57BL/6 and DBA/2 mice acquire activated/memory T cells with age
 
Fas expression on B and T cells is not altered with age
Zhou et al. reported a decline in Fas levels and function in aged mice. However, other reports suggest no change or even increases in Fas levels in aged humans and mice (2124). As illustrated in Fig. 7Go(B), there was no decrease in Fas expression on T or B cells from middle (9–16 months) or old (17–24 months) VH3H9 and Tg BALB/c or C57BL/6 mice, contrary to the findings of Zhou et al. As reported by Wakikawa et al., Fas expression was elevated by ~46 ± 14% (P <= 0.0001) on splenic T cells from aged mice. Additionally, a larger proportion of Fashi splenic B cells was present in aged versus young animals (5.4 ± 5.2 versus 1 ± 0.8% P <= 0.001). These cells may be GC B cells, which express high levels of Fas (64). In support of this, significant increases in the number of PNA+ B cells were also observed in aged mice (young = 1.7 ± 0.6 and aged = 3.8 ± 0.9 P = 0.005) (data not shown). No other age-associated changes in the following B cell developmental or activation markers (B220, CD22, CD23, CD44, CD62L, MHC class II or cell size) were consistently detected with age in any of the strains tested (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Relative to young, aged humans and mice are characterized by a diminished ability to produce antibodies to foreign antigen and a concurrent increase in autoantibody production (14,912). This increased production of autoantibodies, including those directed toward nuclear antigens such as DNA, suggests that the ability to maintain tolerance to self is disrupted with age. To follow the fate of autoreactive B cells in aged mice, we have used the VH3H9 Tg to track anti-dsDNA B cells in vivo. Previously, we have established that anti-dsDNA B cells are actively regulated in young adult VH3H9 BALB/c mice. This is evidenced by the absence of serum autoantibodies, and by the developmental arrest and follicular exclusion of the anti-dsDNA B cells (31). In this study, we have shown that the presence of the VH3H9 Tg did not alter the kinetics of seroconversion in aged mice; therefore, we have used VH3H9 transgenic mice as a model for following the fate of ANA B cells in aged mice. Using this model system, we show that the regulation of anti-dsDNA B cells breaks down in aged VH3H9 mice. In a majority of aged VH3H9 mice, anti-dsDNA B cells no longer line up at the T–B interface, but rather localize in the B cell follicle. This change in splenic localization coincides with a more mature phenotype of the B cells.

The changes in localization and surface phenotype of the anti-dsDNA B cells in aged VH3H9 mice are strikingly similar to those reported for anti-dsDNA B cells in autoimmune-prone MRL-lpr/lpr mice, in which the anti-dsDNA B cells are also mature and in the follicle (33). Importantly, these changes in MRL-lpr/lpr mice precede the appearance of the anti-dsDNA antibodies in their serum. It is possible that in both aging and MRL-lpr/lpr mice a relocation of the autoreactive Ig{lambda}+ B cells into the follicle and a change in their maturation state may be necessary steps for their progression to ASC. Notably, however, and in contrast to MRL-lpr/lpr mice, not all aged VH3H9 BALB/c mice have Ig{lambda}+ anti-dsDNA antibodies in their serum. Additionally, the incidence of ANA and anti-dsDNA antibodies, the titer of ANA, and the extent of nephritis were all lower in aged mice relative to MRL-lpr/lpr mice.

In aged VH3H9 mice, coincident with the altered localization of anti-dsDNA B cells is the infiltration of CD4+ T cells into the B cell follicle. We find similar changes in splenic architecture in aged non-autoimmune Tg BALB/c and C57BL/6 mice. These alterations in CD4+ T cell localization are like those described for MRL-lpr/lpr mice (33) and mice with disrupted LT{alpha} genes (6567). We have shown that the accumulation of activated/memory T cells in aged mice is not the sole factor in determining splenic architecture disruptions as DBA/2 mice acquire activated T cells with age but not the architectural alterations. Nevertheless, these splenic architecture changes are interesting in light of the fact that aged (14), MRL-lpr/lpr (68) and LT{alpha}–/– (66,67) mice are all reported to be hyporesponsive to at least some antigens. In vivo, disruptions in splenic architecture may have important ramifications for the organization of an immune response to foreign antigen. This could in part explain why aged mice exhibit decreased antibody titers and GC reactions in response to antigen associated with aged mice (1–4,13). Furthermore, these disruptions in architecture may alter the localization and affect the regulation of autoreactive B cell populations in both aged and autoimmune-prone mice.


    Acknowledgments
 
The authors wish to thank Hamid Bassiri, Michele Lutz and Dr Lisa Spain for critical reading of the manuscript, Michael Gee for advice on statistical analysis, Dr Clayton Buck for use of his cryostat and microscope, Dr Warren Pear and Dr Lisa Spain for their contribution of aged mice, and Katie Potts, Carrie Sokol and Deepa Kurian for technical assistance. Services provided by The Wistar Institute staff were supported by Core grant no. CA10815, and by grants from the National Institutes of Health (5RO1 AI32137-06), the Arthritis Foundation and the Pew Charitable Trust to J. E. A. E.-B. is supported by a training program in rheumatic diseases (5T3Z AR07442-13), L. M.-N. by a Wistar Training Grant (CA-09171), S. S. by the Medical Scientist Training Program Grant (5T-32GM-07170), M. P. M. by a George M. O'Brien Kidney and Urological Research Center Grant (DK45191) and individual PHS Awards (DK33694 and DK53088), and M. P. C. by NIH grant no. R03-AG15623 and the Lucille P. Markey Charitable Trust.


    Abbreviations
 
ANA anti-nuclear antibodies
AP alkaline phosphatase
ASC antibody-secreting cell
BM bone marrow
ds double-stranded
GC germinal center
H & E hematoxylin & eosin
HN homogeneous nuclear
HRP horseradish peroxidase
MFI mean fluorescence intensity
PALS periarteriolar lymphoid sheath
PE phycoerythrin
SLE systemic lupus erythematosus
Tg transgene

    Notes
 
Transmitting editor: C. Paige

Received 1 October 1999, accepted 25 January 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Goidl, E. A., Innes, J. B. and Weksler, M. E. 1976. Immunological studies of aging. II. Loss of IgG and high avidity plaque-forming cells and increased suppressor cell activity in aging mice. J. Exp. Med. 144:1037.[Abstract]
  2. Makinodan, T. and Kay, M. M. 1980. Age influence on the immune system. Adv. Immunol. 29:287.[Medline]
  3. Schwab, R., Walters, C. A. and Weksler, M. E. 1989. Host defense mechanisms and aging. Semin. Oncol. 16:20.[ISI][Medline]
  4. Bovbjerg, D. H., Kim, Y. T., Schwab, R., Schmitt, K., DeBlasio, T. and Weksler, M. E. 1991. `Cross-wiring' of the immune response in old mice: increased autoantibody response despite reduced antibody response to nominal antigen. Cell. Immunol. 135:519.[ISI][Medline]
  5. Weksler, M. E. and Hutteroth, T. H. 1974. Impaired lymphocyte function in aged humans. J. Clin. Invest. 53:99.[ISI][Medline]
  6. Kishimoto, S., Tomino, S., Mitsuya, H., Fujiwara, H. and Tsuda, H. 1980. Age-related decline in the in vitro and in vivo syntheses of anti-tetanus toxoid antibody in humans. J. Immunol. 125:2347.[Free Full Text]
  7. Burns, E. A., Lum, L. G., Seigneuret, M. C., Giddings, B. R. and Goodwin, J. S. 1990. Decreased specific antibody synthesis in old adults: decreased potency of antigen-specific B cells with aging. Mech. Ageing Dev. 53:229.[ISI][Medline]
  8. Miller, R. A. 1991. Aging and immune function. Int. Rev. Cytol. 124:187.[ISI][Medline]
  9. Rowley, M. J., Buchanan, H. and Mackay, I. R. 1968. Reciprocal change with age in antibody to extrinsic and intrinsic antigens. Lancet ii:24.
  10. Teague, P. O., Friou, G. J. and Myers, L. L. 1968. Anti-nuclear antibodies in mice. I. Influence of age and possible genetic factors on spontaneous and induced responses. J. Immunol. 101:791.[ISI][Medline]
  11. Hallgren, H. M., Buckley, C. E. d., Gilbertsen, V. A. and Yunis, E. J. 1973. Lymphocyte phytohemagglutinin responsiveness, immunoglobulins and autoantibodies in aging humans. J. Immunol. 111:1101.[ISI][Medline]
  12. Welch, M. J., Fong, S., Vaughan, J. and Carson, D. 1983. Increased frequency of rheumatoid factor precursor B lymphocytes after immunization of normal adults with tetanus toxoid. Clin. Exp. Immunol. 51:299.[ISI][Medline]
  13. Yang, X., Stedra, J. and Cerny, J. 1996. Relative contribution of T and B cells to hypermutation and selection of the antibody repertoire in germinal centers of aged mice. J. Exp. Med. 183:959.[Abstract]
  14. Lerner, A., Yamada, T. and Miller, R. A. 1989. Pgp-1hi T lymphocytes accumulate with age in mice and respond poorly to concanavalin A. Eur. J. Immunol. 19:977.[ISI][Medline]
  15. Ernst, D. N., Hobbs, M. V., Torbett, B. E., Glasebrook, A. L., Rehse, M. A., Bottomly, K., Hayakawa, K., Hardy, R. R. and Weigle, W. O. 1990. Differences in the expression profiles of CD45RB, Pgp-1, and 3G11 membrane antigens and in the patterns of lymphokine secretion by splenic CD4+ T cells from young and aged mice. J. Immunol. 145:1295.[Abstract/Free Full Text]
  16. Nagelkerken, L., Hertogh-Huijbregts, A., Dobber, R. and Drager, A. 1991. Age-related changes in lymphokine production related to a decreased number of CD45RBhi CD4+ T cells. Eur. J. Immunol. 21:273.[ISI][Medline]
  17. Utsuyama, M., Hirokawa, K., Kurashima, C., Fukayama, M., Inamatsu, T., Suzuki, K., Hashimoto, W. and Sato, K. 1992. Differential age-change in the numbers of CD4+CD45RA+ and CD4+CD29+ T cell subsets in human peripheral blood. Mech. Ageing Dev. 63:57.[ISI][Medline]
  18. Ernst, D. N., Weigle, W. O., Noonan, D. J., McQuitty, D. N. and Hobbs, M. V. 1993. The age-associated increase in IFN-gamma synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression. J. Immunol. 151:575.[Abstract/Free Full Text]
  19. Linton, P. J., Haynes, L., Klinman, N. R. and Swain, S. L. 1996. Antigen-independent changes in naive CD4 T cells with aging. J. Exp. Med. 184:1891.[Abstract]
  20. Trauth, B. C., Klas, C., Peters, A. M., Matzku, S., Moller, P., Falk, W., Debatin, K. M. and Krammer, P. H. 1989. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245:301.[ISI][Medline]
  21. Zhou, T., Edwards, C. K. r. and Mountz, J. D. 1995. Prevention of age-related T cell apoptosis defect in CD2-fas-transgenic mice [see comments]. J. Exp. Med. 182:129.[Abstract]
  22. Phelouzat, M. A., Laforge, T., Arbogast, A., Quadri, R. A., Boutet, S. and Proust, J. J. 1997. Susceptibility to apoptosis of T lymphocytes from elderly humans is associated with increased in vivo expression of functional Fas receptors. Mech. Ageing Dev. 96:35.[ISI][Medline]
  23. Wakikawa, A., Utsuyama, M. and Hirokawa, K. 1997. Altered expression of various receptors on T cells in young and old mice after mitogenic stimulation: a flow cytometric analysis. Mech. Ageing Dev. 94:113.[ISI][Medline]
  24. Aggarwal, S. and Gupta, S. 1998. Increased apoptosis of T cell subsets in aging humans: altered expression of Fas (CD95), Fas ligand, Bcl-2, and Bax. J. Immunol. 160:1627.[Abstract/Free Full Text]
  25. Flurkey, K., Stadecker, M. and Miller, R. A. 1992. Memory T lymphocyte hyporesponsiveness to non-cognate stimuli: a key factor in age-related immunodeficiency. Eur. J. Immunol. 22:931.[ISI][Medline]
  26. Patel, H. R. and Miller, R. A. 1992. Age-associated changes in mitogen-induced protein phosphorylation in murine T lymphocytes. Eur. J. Immunol. 22:253.[ISI][Medline]
  27. Zharhary, D. 1988. Age-related changes in the capability of the bone marrow to generate B cells. J. Immunol. 141:1863.[Abstract/Free Full Text]
  28. Riley, R. L., Kruger, M. G. and Elia, J. 1991. B cell precursors are decreased in senescent BALB/c mice, but retain normal mitotic activity in vivo and in vitro. Clin. Immunol. Immunopathol. 59:301.[ISI][Medline]
  29. Stephan, R. P., Sanders, V. M. and Witte, P. L. 1996. Stage-specific alterations in murine B lymphopoiesis with age. Int. Immunol. 8:509.[Abstract]
  30. Kline, G. H., Hayden, T. A. and Klinman, N. R. 1999. B cell maintenance in aged mice reflects both increased B cell longevity and decreased B cell generation. J. Immunol. 162:3342.[Abstract/Free Full Text]
  31. Mandik-Nayak, L., Bui, A., Noorchashm, H., Eaton, A. and Erikson, J. 1997. Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T–B interface of the splenic follicle. J. Exp. Med. 186:1257.[Abstract/Free Full Text]
  32. Erikson, J., Radic, M. Z., Camper, S. A., Hardy, R. R., Carmack, C. and Weigert, M. 1991. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature 349:331.[ISI][Medline]
  33. Mandik-Nayak, L., Seo, S., Sokol, C., Potts, K. M., Bui, A. and Erikson, J. 1999. MRL-lpr/lpr mice exhibit a defect in maintaining developmental arrest and follicular exclusion of anti-double-stranded DNA B cells. J. Exp. Med. 189:1799.[Abstract/Free Full Text]
  34. Slater, N. G., Cameron, J. S. and Lessof, M. H. 1976. The Crithidia luciliae kinetoplast immunofluorescence test in systemic lupus erythematosus. Clin. Exp. Immunol. 25:480.[ISI][Medline]
  35. Chan, O., Madaio, M. P. and Shlomchik, M. J. 1997. The roles of B cells in MRL/lpr murine lupus. Ann. NY Acad. Sci. 815:75.[ISI][Medline]
  36. Jacob, J., Kassir, R. and Kelsoe, G. 1991. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173:1165.[Abstract]
  37. Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D. and Hayakawa, K. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213.[Abstract]
  38. Gueret, R., Grandien, A., Andersson, J., Coutinho, A., Radl, J. and Weksler, M. E. 1993. Evidence for selective pressure in the appearance of monoclonal immunoglobulins during aging: studies in M54 mu-transgenic mice. Eur. J. Immunol. 23:1735.[ISI][Medline]
  39. Ten Veen, J. H. and Feltkamp, T. E. W. 1972. Studies on drug induced lupus erythematosus in mice. I. Drug induced antinuclear antibodies (ANA). Clin. Exp. Immunol. 11:265.[ISI][Medline]
  40. Teague, P. O. 1974. Spontaneous autoimmunity and involution of the lymphoid system. Fedn Proc. 33:2051.
  41. Tan, E. M. 1989. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv. Immunol. 44:93.[ISI][Medline]
  42. Eisenberg, R. A., Winfield, J. B. and Cohen, P. L. 1982. Subclass restriction of anti-Sm antibodies in MRL mice. J. Immunol. 129:2146.[Abstract/Free Full Text]
  43. Shlomchik, M., Mascelli, M., Shan, H., Radic, M. Z., Pisetsky, D., Marshak-Rothstein, A. and Weigert, M. 1990. Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation. J. Exp. Med. 171:265.[Abstract]
  44. Radic, M. Z., Mascelli, M. A., Erikson, J., Shan, H. and Weigert, M. 1991. Ig H and L chain contributions to autoimmune specificities. J. Immunol. 146:176.[Abstract/Free Full Text]
  45. Roark, J. H., Kuntz, C. L., Nguyen, K. A., Mandik, L., Cattermole, M. and Erikson, J. 1995. B cell selection and allelic exclusion of an anti-DNA Ig transgene in MRL-lpr/lpr mice. J. Immunol. 154:4444.[Abstract/Free Full Text]
  46. Bell, R. H., Borjesson, B. S., Wolf, P. L., Fernandez-Cruz, L., Brimm, J. E., Lee, S., Sayers, H. J. and Orloff, M. J. 1984. Quantitative morphological studies of aging changes in the kidney of the Lewis rat. Ren. Physiol. 7:176.[ISI][Medline]
  47. Owen, R. A. and Heywood, R. 1986. Age-related variations in renal structure and function in Sprague-Dawley rats. Toxicol. Pathol. 14:158.
  48. Hayashi, Y., Utsuyama, M., Kurashima, C. and Hirokawa, K. 1989. Spontaneous development of organ-specific autoimmune lesions in aged C57BL/6 mice. Clin. Exp. Immunol. 78:120.[ISI][Medline]
  49. Tan, E. M. 1982. Autoantibodies to nuclear antigens (ANA): their immunobiology and medicine. Adv. Immunol. 33:167.[ISI][Medline]
  50. Raff, M. C., Owen, J. J., Cooper, M. D., Lawton, A. R. d., Megson, M. and Gathings, W. E. 1975. Differences in susceptibility of mature and immature mouse B lymphocytes to anti-immunoglobulin-induced immunoglobulin suppression in vitro. Possible implications for B-cell tolerance to self. J. Exp. Med. 142:1052.[Abstract]
  51. Sidman, C. L. and Unanue, E. R. 1975. Receptor-mediated inactivation of early B lymphocytes. Nature 257:149.[ISI][Medline]
  52. Goodnow, C. C., Crosbie, J., Adelstein, S., Lavoie, T. B., Smith-Gill, S. J., Brink, R. A., Pritchard-Briscoe, H., Wotherspoon, J. S., Loblay, R. H., Raphael, K., et al. 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676.[ISI][Medline]
  53. Cooke, M. P., Heath, A. W., Shokat, K. M., Zeng, Y., Finkelman, F. D., Linsley, P. S., Howard, M. and Goodnow, C. C. 1994. Immunoglobulin signal transduction guides the specificity of B cell–T cell interactions and is blocked in tolerant self-reactive B cells. J. Exp. Med. 179:425.[Abstract]
  54. Hartley, S. B., Cooke, M. P., Fulcher, D. A., Harris, A. W., Cory, S., Basten, A. and Goodnow, C. C. 1993. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell 72:325.[ISI][Medline]
  55. Nguyen, K. A., Mandik, L., Bui, A., Kavaler, J., Norvell, A., Monroe, J. G., Roark, J. H. and Erikson, J. 1997. Characterization of anti-single-stranded DNA B cells in a non-autoimmune background. J. Immunol. 159:2633.[Abstract]
  56. Roark, J. H., Bui, A., Nguyen, K. A., Mandik, L. and Erikson, J. 1997. Persistence of functionally compromised anti-double-stranded DNA B cells in the periphery of non-autoimmune mice. Int. Immunol. 9:1615.[Abstract]
  57. Takahashi, K., Kozono, Y., Waldschmidt, T. J., Berthiaume, D., Quigg, R. J., Baron, A. and Holers, V. M. 1997. Mouse complement receptors type 1 (CR1;CD35) and type 2 (CR2;CD21): expression on normal B cell subpopulations and decreased levels during the development of autoimmunity in MRL/lpr mice. J. Immunol. 159:1557.[Abstract]
  58. Smith, K. G., Hewitson, T. D., Nossal, G. J. and Tarlinton, D. M. 1996. The phenotype and fate of the antibody-forming cells of the splenic foci. Eur. J. Immunol. 26:444.[ISI][Medline]
  59. Teuscher, C., Smith, S. M., Goldberg, E. H., Shearer, G. M. and Tung, K. S. 1985. Experimental allergic orchitis in mice. I. Genetic control of susceptibility and resistance to induction of autoimmune orchitis. Immunogenetics 22:323.[ISI][Medline]
  60. Zheng, B., Han, S. and Kelsoe, G. 1996. T helper cells in murine germinal centers are antigen-specific emigrants that downregulate Thy-1. J. Exp. Med. 184:1083.[Abstract]
  61. Brocker, T., Gulbranson-Judge, A., Flynn, S., Riedlinger, M., Raykundalia, C. and Lane, P. 1999. CD4 T cell traffic control: in vivo evidence that ligation of OX40 on CD4 T cell by OX40-ligand expressed on dendritic cells leads to the accumulation of CD4 T cells in B follicles. Eur. J. Immunol. 29:1610.[ISI][Medline]
  62. Gulbranson-Judge, A. and MacLennan, I. 1996. Sequential antigen-specific growth of T cells in the T zones and follicles in response to pigeon cytochrome c. Eur. J. Immunol. 26:1830.[ISI][Medline]
  63. Garside, P., Ingulli, E., Merica, R. R., Johnson, J. G., Noelle, R. J. and Jenkins, M. K. 1998. Visualization of specific B and T lymphocyte interactions in the lymph node. Science 281:96.[Abstract/Free Full Text]
  64. Mandik, L., Nguyen, K. A. and Erikson, J. 1995. Fas receptor expression on B-lineage cells. Eur. J. Immunol. 25:3148.[ISI][Medline]
  65. De Togni, P., Goellner, J., Ruddle, N. H., Streeter, P. R., Fick, A., Mariathasan, S., Smith, S. C., Carlson, R., Shornick, L. P., Strauss-Schoenberger, J., et al. 1994. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin [see comments]. Science 264:703.[ISI][Medline]
  66. Banks, T. A., Rouse, B. T., Kerley, M. K., Blair, P. J., Godfrey, V. L., Kuklin, N. A., Bouley, D. M., Thomas, J., Kanangat, S. and Mucenski, M. L. 1995. Lymphotoxin-alpha-deficient mice. Effects on secondary lymphoid organ development and humoral immune responsiveness. J. Immunol. 155:1685.[Abstract]
  67. Koni, P. A., Sacca, R., Lawton, P., Browning, J. L., Ruddle, N. H. and Flavell, R. A. 1997. Distinct roles in lymphoid organogenesis for lymphotoxins alpha and beta revealed in lymphotoxin beta-deficient mice. Immunity 6:491.[ISI][Medline]
  68. Creighton, W. D., Katz, D. H. and Dixon, F. J. 1979. Antigen-specific immunocompetency, B cell function, and regulatory helper and suppressor T cell activities in spontaneously autoimmune mice. J. Immunol. 123:2627.[ISI][Medline]
  69. Lynch, F. and Ceredig, R. 1989. Mouse strain variation in Ly-24 (Pgp-1) expression by peripheral T cells and thymocytes: implications for T cell differentiation. Eur. J. Immunol. 19:223.[ISI][Medline]