Circulating autoantibodies against dsDNA and chromatin are a characteristic of systemic lupus erythematosus in humans and many mouse models of this disease. B cells expressing these autoantibodies are normally regulated in nonautoimmune-prone mice but are induced to secrete Abs following T cell help. Likewise, anti-chromatin autoantibody production is T cell-dependent in Fas/Fas ligand (FasL)-deficient (lpr/lpr or gld/gld) mice. In this study, we demonstrate that Th2 cells promote anti-chromatin B cell survival and autoantibody production in vivo. FasL influences the ability of Th2 cells to help B cells, as Th2-gld/gld cells support higher titers of anti-chromatin Abs than their FasL-sufficient counterparts and promote anti-chromatin B cell participation in germinal centers. Th1 cells induce anti-chromatin B cell germinal centers regardless of FasL status; however, their ability to stimulate anti-chromatin Ab production positively correlates with their level of IFN-γ production. This distinction is lost if FasL-deficient T cells are used: Th1-gld/gld cells promote significant titers of anti-chromatin Abs regardless of IFN-γ production levels. Thus, FasL from effector T cells plays an important role in determining the fate of anti-chromatin B cells.

Antibodies that bind dsDNA and chromatin are a serologic hallmark of systemic lupus erythematosus and are found in several murine models of systemic lupus erythematosus (1). Using the VH3H9 Ig transgenic (Tg) 3 model, a population of anti-chromatin B cells (VH3H9/Vλ1) has been characterized in both healthy and autoimmune mice (2, 3, 4, 5, 6, 7). In healthy mice, these anti-chromatin B cells persist in the periphery but their Abs are undetected (2, 5). They have a decreased life span, are predominantly developmentally arrested and activated, and localize to the edges of the B cell follicles near the T cell areas in the spleen (5, 8). We hypothesize that this phenotype is a consequence of chronic exposure to Ag, in the absence of T cell help (9, 10, 11, 12, 13).

To dissect the responses of anti-chromatin B cells to CD4+ Th cells, we established an in vivo model of cognate interaction between these two cell types. Mice engineered to express the neoself Ag hemagglutinin (HA) on MHC class II-bearing cells (including B cells) were mated to VH3H9 Ig Tg mice (14, 15). Anti-HA CD4+ T cells from TCR Tg mice and HA-expressing anti-chromatin B cells were then transferred together into a third-party recipient mouse and their fates tracked (16, 17). Using this strategy, we have demonstrated that anti-chromatin B cells from healthy mice respond to CD4+ Th cells in vivo by producing autoantibodies (16). These findings contrast with those obtained from the hen-egg lysozyme model of B cell tolerance (18, 19), where autoreactive anti-hen-egg lysozyme B cells were shown to be resistant to CD4+ T cell help (20, 21, 22, 23). Importantly, Fas/ Fas ligand (FasL) interactions mediated this resistance (20, 21, 24).

Both Th1 and Th2 cells can help nonautoreactive B cells to produce Abs (25, 26, 27), although Th2 cells appear more efficient in this regard (28, 29, 30, 31). Th1 cells reportedly express higher levels of FasL than Th2 cells (32, 33, 34, 35), which could modulate T-B interactions by either limiting the availability of Th cells (32, 33, 35) and/or by direct killing of the autoreactive B cells (20, 21). Furthermore, Th1 cells may be more susceptible to Fas-mediated death even in cases where they express comparable levels of FasL (32, 33, 35).

Once activated, B cells can follow a number of differentiation pathways–toward short-lived Ab-forming cells (AFCs), memory B cells, or long-lived AFCs. Although much has been learned about the transcription factors that govern the fate decisions of B cells (36), less is known about the precise cues that determine these decisions. Given the critical role that CD4+ T cells play in autoantibody production, and the seemingly contradictory data on autoreactive B cell responses to T cell help (20, 21, 22, 23), we have investigated the impact of Th1 and Th2 cells, with and without FasL, on anti-chromatin B cells. We report here the differential capabilities of T effector cells to influence anti-chromatin B cells in vivo, and suggest that FasL may play a role in controlling the differentiation pathways and magnitude of the autoreactive B cell response.

TS1 BALB/c (FasL-sufficient or -deficient) and VH3H9 Tg/HACII/Igκ−/− mice were bred in specific-pathogen-free conditions at The Wistar Institute under the approval and supervision of the Institutional Animal Care and Use Committee (IACUC), and genotyped as described (2, 6, 16, 17, 37). Similarly, site-directed VH3H9 Tg BALB/c mice in which the VH3H9 Tg was targeted to the JH locus (Refs. 38, 39, 40 ; generously provided by Dr. M. Weigert (University of Chicago, Chicago, IL) were bred to produce site-directed VH3H9 Tg/HACII/Igκ−/− mice. CB17 mice were purchased from Charles River Laboratories. Only young (6–12 wk old) TS1 BALB/c-gld/gld mice were used. All other mice were used at 6–16 wk, and both genders were used.

TS1 BALB/c or TS1 BALB/c-gld/gld lymph nodes were depleted (>90%) of CD8+ cells using anti-CD8 Dynalbeads (Dynaltech). A total of 0.5 × 106 CD8-depleted lymphocytes were then cultured for Th1/Th2 deviation as previously described (41). rIL-12 was a generous gift from Dr. G. Trinchieri (National Institutes of Health, Bethesda, MD) or was purchased from R&D Biosystems or PeproTech. Cells received fresh media containing IL-2 at days 3 and 5, and were rested in the absence of IL-2 at day 7. At day 9, cells were harvested and an aliquot cultured with PMA and ionomycin (Sigma-Aldrich) in the presence of brefeldin A (Cytofix/Cytoperm kit; BD Pharmingen) for another 4–6 h. Cells were stained for CD4 expression, fixed, permeabilized, and stained for intracellular cytokines, using anti-IFN-γ-FITC, anti-IL-4-PE, and/or anti-IL-10-PE (BD Pharmingen) (42). The cells that were not restimulated with PMA/ionomycin were purified by centrifugation with Lympholyte M (Cedarlane Laboratories) before injection.

A total of 5–10 × 106 Th1- or Th2-deviated CD4+ cells from in vitro cultures were suspended in sterile PBS with 1000 hemagglutinating units (43) of purified PR8 influenza virus (15) and injected i.v.

Splenocytes from VH3H9 Tg/HACII/Igκ−/− or site-directed VH3H9 Tg/HACII/Igκ−/− mice were depleted of RBC and an aliquot was stained by flow cytometry to determine the frequency of anti-chromatin B cells (B220+ Igλ1+). CB17 recipient mice (allotype Igb) were injected with spleen preparations containing 4–10 × 106 anti-chromatin B cells (allotype Iga). Control mice received B cells without previous injection of Th cells or virus.

Mice were i.p. injected with 250 μg of purified anti-CD154 mAb (MR1) (a kind gift of Dr. R. Noelle, Dartmouth Medical School, Lebanon, NH) on the same day as B cell transfer, and 3 days afterward.

Chromatin (a generous gift of Dr. M. Monestier, Temple University, Philadelphia, PA) was diluted to 2 μg/ml and plated overnight as described (44). ELISAs were done as described (45) with the following modifications: the block used was 1% BSA/PBS/azide, developing Abs were anti-Igλ1, anti-IgMa, anti-IgG1, or anti-IgG2aa (all biotinylated, BD Pharmingen), followed by avidin-alkaline phosphatase (AP; Southern Biotechnology Associates). Plates were developed for 14–18 h with ImmunoPure PNPP (Pierce) as the substrate. OD values were recorded and background values were subtracted out (background was defined as the OD value generated by a hybridoma supernatant of irrelevant specificity, typically ∼0.07). All developing Abs were allotype-marked (Iga) except for IgG1, (due to poor sensitivity of the anti-IgG1a Ab in ELISAs). Although the IgG1 reagent was not allotype-marked, sera from uninjected CB17 mice showed no significant staining above background for IgG1 anti-chromatin Abs. Points derived from the linear range of the ELISAs were used for generating graphs. For studies examining whether long-lived plasma cells are generated when T cell help is provided, mice were serially bled and their sera tested by ELISA. When anti-chromatin OD values returned to baseline levels for a mouse, they were no longer bled or tested.

A total of 0.5–1 × 106 splenocytes were surface stained (46) using the following Abs: anti-B220-FITC (RA3-6B2), anti-CD4-PE (GK1.5), anti-IgMa-PE or -bio (DS-1), anti-Igλ1-bio (R11-153), IgDa-bio (217–170), IgG1a-bio (10.9), IgG2aa-bio (8.3) (BD Pharmingen), and 6.5-biotin (grown as supernatant and biotinylated). Biotinylated peanut agglutinin (PNA; Vector Laboratories) was also used to mark germinal center (GC) B cells.

The frequency of IgMa+Igλ1+ B cells or CD4+6.5+ T cells in the spleen was determined by flow cytometry and multiplied by the total number of live splenocytes to determine the absolute number of cells. The percent recovery of transferred B or T cells was determined by dividing the absolute number of cells recovered by the number of cells injected.

Spleens were frozen, sectioned, and stained (5) using the following Abs: anti-CD4-bio (GK1.5), anti-CD22-FITC or -bio (Cy34.1), anti-IgMa-FITC (DS-1), anti-IgG1a-bio (10.9), anti-IgG2aa-bio (8.3) (BD Pharmingen), and/or PNA-bio (Vector Laboratories). Secondary reagents were anti-FITC-AP, anti-FITC-HRP, streptavidin-AP, or streptavidin-HRP (Southern Biotechnology Associates). Developed slides were read by multiple (at least four) investigators without prior knowledge of the experimental condition.

Statistical significance was determined via the unpaired, two sample Student’s t test provided by Microsoft Excel software unless otherwise noted. Significance was ascribed when p < 0.05.

The VH3H9 H chain paired with the Vλ1 L chain generates an Ab that binds dsDNA and chromatin (47, 48). Therefore, the VH3H9 Tg can be used to directly track autoreactive B cells in vivo by staining for endogenous Vλ1. To test the impact of cognate T cell help on anti-chromatin B cells in vivo, HA-bearing anti-chromatin B cells were injected into third-party recipient CB17 mice that had received distinct subsets of anti-HA CD4+ T cells (Fig. 1; Ref. 16).

FIGURE 1.

Experimental protocol. CB17 mice (allotype IgMb) were injected with influenza virus and anti-HA CD4+ T cells (from wild-type or FasL-deficient mice) that were cultured in vitro under Th1 or Th2 conditions. The following day, anti-chromatin B cells (α-chromatin-B); IgMa allotype from either VH3H9/HACII/Igκ−/− or site-directed VH3H9 Tg/HACII/Igκ−/− BALB/c mouse spleens) were injected. Mice were analyzed on day 8.

FIGURE 1.

Experimental protocol. CB17 mice (allotype IgMb) were injected with influenza virus and anti-HA CD4+ T cells (from wild-type or FasL-deficient mice) that were cultured in vitro under Th1 or Th2 conditions. The following day, anti-chromatin B cells (α-chromatin-B); IgMa allotype from either VH3H9/HACII/Igκ−/− or site-directed VH3H9 Tg/HACII/Igκ−/− BALB/c mouse spleens) were injected. Mice were analyzed on day 8.

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Before injection, anti-HA CD4+ T cells from FasL-sufficient or -deficient mice were polarized into either the Th1 or Th2 subset (Fig. 2). Th2 cells from FasL-sufficient and -deficient mice produced IL-4 (20–40% of cells) and IL-10 (3–10% of cells) (Fig. 2). In addition, a small population of Th2-gld/gld cells produced IFN-γ (Fig. 2, 2.5% of cells vs 0.53% of cells from FasL-sufficient mice, p < 0.01).

FIGURE 2.

Cytokine production by in vitro-deviated cells. Th1 or Th2 cells were generated in vitro from either TS1 Tg BALB/c or TS1 Tg BALB/c-gld/gld cells and tested for production of IFN-γ and either IL-4 (top) or IL-10 (bottom). The range for cells producing IFN-γ from Th1 cultures was 2–70%. Thus, Th1 cells are grouped into high or low IFN-γ-producing categories. Graphs are representative of n ≥ 4 for each cell type.

FIGURE 2.

Cytokine production by in vitro-deviated cells. Th1 or Th2 cells were generated in vitro from either TS1 Tg BALB/c or TS1 Tg BALB/c-gld/gld cells and tested for production of IFN-γ and either IL-4 (top) or IL-10 (bottom). The range for cells producing IFN-γ from Th1 cultures was 2–70%. Thus, Th1 cells are grouped into high or low IFN-γ-producing categories. Graphs are representative of n ≥ 4 for each cell type.

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Th1-deviated cells derived from wild-type or gld/gld mice produced little or no IL-4 or IL-10, and varied widely according to the amount of IFN-γ they made (Fig. 2). One group of Th1 cells had 40–70% of the cells producing IFN-γ without detectable IL-4 or IL-10, and such cells are termed IFN-γhigh Th1 cells. In contrast, the second group, which also did not make IL-4 or IL-10, yielded fewer IFN-γ-producing cells (2–35%), and less IFN-γ per cell, as visualized by lower intracellular IFN-γ fluorescent intensity (Fig. 2). This second group is termed IFN-γlow Th1 cells. This variation in IFN-γ production proved to be significant in terms of the ability of the T cells to help anti-chromatin B cells (see below) and appeared dependent upon the source of rIL-12 in cultures. To directly examine the effects of limiting IL-12 on deviated Th cells, cultures were set up in the absence of exogenous rIL-12, but in the presence of anti-IL-4 Ab to prevent acquisition of a Th2-like phenotype (cells resulting from such cultures are termed IFN-γlowΔ cells). A low frequency of CD4+ T cells (2–8%) from these cultures produced IFN-γ, with low levels of IFN-γ per cell, and <1% produced IL-4 or IL-10. Both the IFN-γhigh and IFN-γlow intracellular cytokine levels are within the range of those reported by others for Th1-deviated cultures (41, 42, 49, 50).

Th2 cells promoted anti-chromatin autoantibodies from the transferred B cells (Fig. 3). Autoantibody production from mice given Th1 cells was variable and correlated (p < 0.01) with the level of IFN-γ produced by the Th1 cells before their injection. IFN-γhigh Th1 cells induced levels of anti-chromatin autoantibodies similar to those seen in Th2 recipients, whereas levels in recipients of IFN-γlow and IFN-γlowΔ Th1 cells were not above the baseline (Fig. 3). The observation that IFN-γ levels affect the ability of Th1 cells to help B cells may resolve a controversy regarding the ability of Th1 cells to serve as helpers (25, 26, 27, 28, 29, 30, 31), as laboratories using distinct in vitro deviation protocols may generate differing frequencies of IFN-γ-producing Th1 cells.

FIGURE 3.

Th2 cells and IFN-γhigh Th1 cells induce anti-chromatin autoantibody production. Sera were assayed by ELISA for IgMa anti-chromatin Abs (serum dilution 1/100). Bar graphs show mean OD value for each experimental condition, ±SEM; ∗, significant difference (p < 0.05). Th1 recipients are divided according to whether they received IFN-γhigh or IFN-γlow Th1 cells. Also shown are results from mice that received T cells from either FasL-sufficient or -deficient mice, cultured with anti-IL-4 and no exogenous rIL-12 (marked as IFN-γlowΔ). All graphs with an average above 1.0 are significantly different from the condition with B cells alone. Ø, B cells alone. Sample sizes: B cells alone, n = 4; Th2, n = 10; Th2 + MR1, n = 3; IFN-γhigh Th1, n = 7; IFN-γhigh Th1 + MR1, n = 3; IFN-γlow Th1, n = 7; IFN-γlowΔ Th1, n = 6; IFN-γhigh Th1-gld/gld, n = 3; IFN-γlow Th1-gld/gld, n = 3; IFN-γlowΔ Th1-gld/gld, n = 3.

FIGURE 3.

Th2 cells and IFN-γhigh Th1 cells induce anti-chromatin autoantibody production. Sera were assayed by ELISA for IgMa anti-chromatin Abs (serum dilution 1/100). Bar graphs show mean OD value for each experimental condition, ±SEM; ∗, significant difference (p < 0.05). Th1 recipients are divided according to whether they received IFN-γhigh or IFN-γlow Th1 cells. Also shown are results from mice that received T cells from either FasL-sufficient or -deficient mice, cultured with anti-IL-4 and no exogenous rIL-12 (marked as IFN-γlowΔ). All graphs with an average above 1.0 are significantly different from the condition with B cells alone. Ø, B cells alone. Sample sizes: B cells alone, n = 4; Th2, n = 10; Th2 + MR1, n = 3; IFN-γhigh Th1, n = 7; IFN-γhigh Th1 + MR1, n = 3; IFN-γlow Th1, n = 7; IFN-γlowΔ Th1, n = 6; IFN-γhigh Th1-gld/gld, n = 3; IFN-γlow Th1-gld/gld, n = 3; IFN-γlowΔ Th1-gld/gld, n = 3.

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CD40-CD154 interactions play a vital role in Th cell activity, including autoimmune settings (51, 52, 53, 54). Likewise, the blocking anti-CD154 mAb (MR1) abrogated anti-chromatin Ab production stimulated by either Th2 or IFN-γhigh Th1 cells (Fig. 3).

Injection of Th2-deviated gld/gld cells enhanced the production of anti-chromatin Abs in recipient mice compared with the titers observed with Th2 cells derived from wild-type mice (Fig. 3). There was no difference in anti-chromatin Ab titers between IFN-γhigh Th1 cells and IFN-γhigh Th1-gld/gld cells (Fig. 3, p = 0.34), whereas IFN-γlow Th1-gld/gld T cells promoted higher titers of anti-chromatin Abs than their IFN-γlow FasL-sufficient counterparts (Fig. 3).

The site-directed VH3H9 Tg was used to monitor anti-chromatin isotype switching. CB17 mice that received Th2 cells or IFN-γhigh Th1 cells and site-directed Tg anti-chromatin B cells had detectable titers of Igλ1 anti-chromatin Abs (Fig. 4,A). These Abs included not only IgMa Abs (Fig. 4,B) but also isotype-switched Abs (Fig. 4, C and D). IFN-γhigh Th1 cell help resulted in higher titers of IgG2aa autoantibodies than mice given either Th2 cells or B cells alone. Th2 recipients produced high levels of IgG1 anti-chromatin Abs and low, but also significant (relative to recipients of B cells alone), titers of IgG2aa anti-chromatin Abs. Like what was observed using the non-site-directed VH3H9 Tg donors, IFN-γlow Th1 cells did not induce anti-chromatin Abs.

FIGURE 4.

Th2 cells and IFN-γhigh Th1 cells induce isotype-switched anti-chromatin Abs. Sera were assayed by ELISA for anti-chromatin Abs for (A) total Igλ1, (B) IgMa, (C) IgG1, and (D) IgG2aa. OD values were recorded at the following serum dilutions for the ELISAs: 1/400 for Igλ1, 1/800 for IgMa, and 1/100 for both IgG1 and IgG2aa. Bar graphs show mean values ± SEM; ∗, significant difference (p < 0.05) between experimental groups. Δ, Significant difference compared with mice given B cells alone. Ø, B cells alone. Sample sizes: B cells alone, n = 10; Th2, n = 7; IFN-γhigh Th1, n = 4; Th2-gld/gld, n = 6; IFN-γhigh Th1-gld/gld, n = 6.

FIGURE 4.

Th2 cells and IFN-γhigh Th1 cells induce isotype-switched anti-chromatin Abs. Sera were assayed by ELISA for anti-chromatin Abs for (A) total Igλ1, (B) IgMa, (C) IgG1, and (D) IgG2aa. OD values were recorded at the following serum dilutions for the ELISAs: 1/400 for Igλ1, 1/800 for IgMa, and 1/100 for both IgG1 and IgG2aa. Bar graphs show mean values ± SEM; ∗, significant difference (p < 0.05) between experimental groups. Δ, Significant difference compared with mice given B cells alone. Ø, B cells alone. Sample sizes: B cells alone, n = 10; Th2, n = 7; IFN-γhigh Th1, n = 4; Th2-gld/gld, n = 6; IFN-γhigh Th1-gld/gld, n = 6.

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Th2-gld/gld cells induced higher titers of Igλ1 anti-chromatin Abs from site-directed VH3H9 Tg B cells than their FasL-sufficient counterparts (Fig. 4). This increase included higher titers of the IgMa and IgG2aa isotypes, but similar levels of the IgG1 isotype. The increased level of IFN-γ production by gld/gld-derived Th2 cells (Fig. 2) may account for the higher IgG2a isotype anti-chromatin Abs (25). In contrast, the absence of functional FasL on IFN-γhigh Th1 cells did not have an effect on the levels of Igλ1 anti-chromatin Abs produced or their isotype distribution (Fig. 4, p ≥ 0.05 for Igλ1, IgMa, or IgG2aa ELISAs).

In the absence of exogenous Th cells, very few anti-chromatin B cells remained 7 days after transfer (Fig. 5), consistent with their rapid in vivo turnover rate (8). Both Th2 and IFN-γhigh Th1 cells supported anti-chromatin B cell recovery and this was dependent upon CD40-CD154 interactions (Fig. 5). FasL-deficient Th2 and IFN-γhigh Th1 cells promoted similar recoveries compared with their wild-type counterparts. In contrast, IFN-γlow Th1 cells derived from gld/gld mice promoted anti-chromatin B cell survival well above that from FasL-sufficient IFN-γlow Th1 cells (Fig. 5).

FIGURE 5.

Recovery of transferred anti-chromatin B cells given T cell help. A, Flow cytometry was used to determine the percent recovery of transferred anti-chromatin B cells (Igλ1+IgMa+ cells). Shown are representative graphs. B, Bar graphs show mean values ± SEM; ∗, significant difference (p < 0.05) between experimental groups. Δ, Significant difference compared with mice given B cells alone. Ø, B cells alone. Sample sizes: B cells alone, n = 12; Th2, n = 16; Th2 + MR1, n = 3; Th2-gld/gld, n = 8; IFN-γhigh Th1, n = 10; IFN-γhigh Th1 + MR1, n = 3; IFN-γlow Th1, n = 10; IFN-γhigh Th1-gld/gld, n = 4; IFN-γlow Th1-gld/gld, n = 6.

FIGURE 5.

Recovery of transferred anti-chromatin B cells given T cell help. A, Flow cytometry was used to determine the percent recovery of transferred anti-chromatin B cells (Igλ1+IgMa+ cells). Shown are representative graphs. B, Bar graphs show mean values ± SEM; ∗, significant difference (p < 0.05) between experimental groups. Δ, Significant difference compared with mice given B cells alone. Ø, B cells alone. Sample sizes: B cells alone, n = 12; Th2, n = 16; Th2 + MR1, n = 3; Th2-gld/gld, n = 8; IFN-γhigh Th1, n = 10; IFN-γhigh Th1 + MR1, n = 3; IFN-γlow Th1, n = 10; IFN-γhigh Th1-gld/gld, n = 4; IFN-γlow Th1-gld/gld, n = 6.

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Immunohistochemistry was used to further examine the differentiation status of the anti-chromatin B cells (Fig. 6). Clusters of extrafollicular anti-chromatin B cells were observed in the spleens of mice given Th2 cells or IFN-γhigh Th1 cells (Fig. 6,A). These cells exhibited substantially higher Ig staining levels than follicular B cells, and expressed CD138 (data not shown), a marker of AFCs. Consistent qualitative differences in the AFCs are apparent, with AFCs in Th2-recipient mice being more tightly clustered in extrafollicular foci (EFF), than those in mice given IFN-γhigh Th1 cells (Fig. 6,A). Anti-chromatin AFCs were not observed in mice that received IFN-γlow Th1 cells, consistent with the absence of anti-chromatin Abs (Fig. 6 B).

FIGURE 6.

Splenic localization of anti-chromatin B cells given T cell help. Mice received deviated Th cells, virus, and anti-chromatin B cells from VH3H9/HACII/κ−/− mice (A and B), or site-directed VH3H9 Tg/HACII/κ−/− mice (C), and were sacrificed 7 days after B cell transfer. Serial spleen sections were stained with Abs to CD22 and either IgMa, IgG1a, IgG2aa, or PNA, as indicated. Pictures are representative of n ≥ 5 for each experimental condition.

FIGURE 6.

Splenic localization of anti-chromatin B cells given T cell help. Mice received deviated Th cells, virus, and anti-chromatin B cells from VH3H9/HACII/κ−/− mice (A and B), or site-directed VH3H9 Tg/HACII/κ−/− mice (C), and were sacrificed 7 days after B cell transfer. Serial spleen sections were stained with Abs to CD22 and either IgMa, IgG1a, IgG2aa, or PNA, as indicated. Pictures are representative of n ≥ 5 for each experimental condition.

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Few GCs are visible in CB17 mice given only anti-chromatin B cells, whereas mice also injected with Th cells and virus developed GCs in at least 50% of the B cell follicles (Fig. 6, A and B) and this was blocked by MR1 (data not shown). Although GCs were observed in Th2 recipients, they do not include the transferred Iga cells and are likely a consequence of endogenous CB17 B cells (Igb) responding to virus. In contrast, recipients of IFN-γhigh Th1 cells have many instances of IgMa cells in B cell follicles, often localizing in GCs (Fig. 6,A, arrows). Strikingly, the few transferred anti-chromatin B cells that persist in IFN-γlow Th1 recipients are primarily associated with GCs (Fig. 6,B). Flow cytometry was used to quantitate the higher frequency of GCs in Th1 recipients (IFN-γhigh and IFN-γlow) compared with Th2 recipients (Fig. 7).

FIGURE 7.

GC phenotype of anti-chromatin B cells. Flow cytometry was used to determine frequency of IgMa+ cells that were PNA+ in mice given T cell help. Bar graphs show mean values and circles represent values from individual mice. ∗, Significant difference (p < 0.05). Sample sizes: Th2, n = 10; Th2-gld/gld, n = 7; IFN-γhigh Th1, n = 10; IFN-γlow Th1, n = 4; IFN-γhigh Th1-gld/gld, n = 4; IFN-γlow Th1-gld/gld, n = 4.

FIGURE 7.

GC phenotype of anti-chromatin B cells. Flow cytometry was used to determine frequency of IgMa+ cells that were PNA+ in mice given T cell help. Bar graphs show mean values and circles represent values from individual mice. ∗, Significant difference (p < 0.05). Sample sizes: Th2, n = 10; Th2-gld/gld, n = 7; IFN-γhigh Th1, n = 10; IFN-γlow Th1, n = 4; IFN-γhigh Th1-gld/gld, n = 4; IFN-γlow Th1-gld/gld, n = 4.

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When FasL-deficient Th2 cells were administered, tight clusters of anti-chromatin AFCs were observed, similar to their FasL-sufficient counterparts (Fig. 6,A). Anti-chromatin B cells from Th2-gld/gld recipients, however, were also present in GCs with a higher frequency compared with mice given FasL-sufficient Th2 cells (Fig. 6,A, arrows, and Fig. 7). Recipients of IFN-γhigh Th1-gld/gld T cells had anti-chromatin GCs as did their wild-type counterparts, but their EFF were even more diffuse. Most strikingly, IFN-γlow Th1-gld/gld cells induced not only anti-chromatin GCs but also AFCs (data summarized in Table I).

Table I.

Fate of transferred anti-chromatin B cells and Th cells

ConditionTh cellsAnti-Chromatin B Cells
Anti-chromatin B cells plus:RecoveryaSerum auto- antibodiesRecoveryaLocalizationa
No exogenous T cells N/Ab − − − 
Th2 ++ AFCs 
Th2-gld/gld +c ++ AFCs GCs 
IFN-γhighTh1 ++ ++ AFCs GCs 
IFN-γlowTh1 − GCs 
IFN-γhighTh1-gld/gld ++ AFCs GCs 
IFN-γlowTh1-gld/gld ++ AFCs GCs 
ConditionTh cellsAnti-Chromatin B Cells
Anti-chromatin B cells plus:RecoveryaSerum auto- antibodiesRecoveryaLocalizationa
No exogenous T cells N/Ab − − − 
Th2 ++ AFCs 
Th2-gld/gld +c ++ AFCs GCs 
IFN-γhighTh1 ++ ++ AFCs GCs 
IFN-γlowTh1 − GCs 
IFN-γhighTh1-gld/gld ++ AFCs GCs 
IFN-γlowTh1-gld/gld ++ AFCs GCs 
a

Data were obtained from analysis at day 8 (7 days after B cell transfer). With regard to cell recovery and serum autoantibody levels, “−”, “+”, and “++” indicate statistically significant differences (p < 0.05).

b

N/A, not applicable.

c

Serum autoantibody titers from Th2-gld/gld recipients were significantly higher than their FasL-sufficient counterparts.

With site-directed VH3H9 Tg mice used as a source of anti-chromatin B cells, in conjunction with T cell help, isotype-switched AFCs were detected. As was observed in the serum, recipients of Th2 cells had anti-chromatin EFF mainly comprised of IgMa and IgG1a cells, with rare IgG2aa cells, whereas recipients of IFN-γhigh Th1 cells have EFF containing both IgMa and IgG2aa but not IgG1a cells (Fig. 6 C). Minimal Iga cells were visualized in recipients of site-directed VH3H9 Tg B cells in the absence of exogenous Th cells (data not shown).

To test the fate of the transferred Th1 or Th2 cells, splenocytes of recipient mice were stained with the 6.5 clonotypic Ab (Fig. 8). A distinct population of CD4+6.5+ cells could be detected in spleens of mice given Th cells, but not in the absence of exogenous Th cells (Fig. 8,A). CD40-CD154 blockade significantly inhibited recovery of both Th2 and IFN-γhigh Th1 cells (Fig. 8,B). Of the CD4+ Th cells, IFN-γhigh Th1 cells displayed the highest level of cell recovery; notably, recovery of these cells was decreased when they were derived from FasL-deficient mice (Fig. 8 B). This suggests that, under some conditions, FasL may provide a positive signal, as was suggested for CD4+ cells in vitro (55).

FIGURE 8.

Th cell recoveries in the spleen. A, Spleen cells were stained with 6.5 (clonotype) and anti-CD4 to determine recovery of transferred Th cells in recipient mice. Plots are representative of n ≥ 10 mice. B, The absolute number of CD4+ clonotype+ Th cells was determined and the percent recovery of transferred cells was plotted. Bar graphs depict mean values ± SEM; ∗, significant difference (p < 0.05). Sample sizes: Th2, n = 14; Th2 + MR1, n = 3; Th2-gld/gld, n = 9; IFN-γhigh Th1, n = 10; Th1 + MR1, n = 3; IFN-γlow Th1, n = 8; IFN-γhigh Th1-gld/gld, n = 4; IFN-γlow Th1-gld/gld, n = 6.

FIGURE 8.

Th cell recoveries in the spleen. A, Spleen cells were stained with 6.5 (clonotype) and anti-CD4 to determine recovery of transferred Th cells in recipient mice. Plots are representative of n ≥ 10 mice. B, The absolute number of CD4+ clonotype+ Th cells was determined and the percent recovery of transferred cells was plotted. Bar graphs depict mean values ± SEM; ∗, significant difference (p < 0.05). Sample sizes: Th2, n = 14; Th2 + MR1, n = 3; Th2-gld/gld, n = 9; IFN-γhigh Th1, n = 10; Th1 + MR1, n = 3; IFN-γlow Th1, n = 8; IFN-γhigh Th1-gld/gld, n = 4; IFN-γlow Th1-gld/gld, n = 6.

Close modal

To determine whether long-lived anti-chromatin plasma cells are generated when T cell help is provided, CB17 mice were injected with IFN-γhigh Th1 or Th2 cells and anti-chromatin B cells from either VH3H9 Tg or site-directed VH3H9 Tg donors, and then serially bled for up to 10 wk. In all mice given Th cells, anti-chromatin Ab titers peaked at days 8–11 and then declined such that by day 36, levels were similar to those in uninjected mice and mice given B cells without exogenous T cells (Table II).

Table II.

T cell-induced anti-chromatin Abs do not persista

ConditionDay 0Day 5–11Day 36+
Anti-chromatin Abs (IgMa   
 B cells alone (n = 4) − − NT 
 IFN-γhighTh1 (n = 5) − − 
 IFN-γhighTh1-gld/gld (n = 6) − − 
 Th2 (n = 4) − − 
 Th2-gld/gld (n = 5) − − 
Site-directed VH3H9 Tg donors-anti-chromatin Abs (Igλ1)    
 B cells alone (n = 4) − − − 
 IFN-γhighTh1 (n = 2) − − 
 IFN-γhighTh1-gld/gld (n = 4) − − 
 Th2 (n = 4) − − 
 Th2-gld/gld (n = 3) − − 
ConditionDay 0Day 5–11Day 36+
Anti-chromatin Abs (IgMa   
 B cells alone (n = 4) − − NT 
 IFN-γhighTh1 (n = 5) − − 
 IFN-γhighTh1-gld/gld (n = 6) − − 
 Th2 (n = 4) − − 
 Th2-gld/gld (n = 5) − − 
Site-directed VH3H9 Tg donors-anti-chromatin Abs (Igλ1)    
 B cells alone (n = 4) − − − 
 IFN-γhighTh1 (n = 2) − − 
 IFN-γhighTh1-gld/gld (n = 4) − − 
 Th2 (n = 4) − − 
 Th2-gld/gld (n = 3) − − 
a

CB17 mice received Th cells, virus, and anti-chromatin B cells from VH3H9 Tg/HACII/κ−/− or site-directed VH3H9 Tg/HACII/κ−/− mice, and were bled at intervals thereafter. IgMa or Igλ1 anti-chromatin Abs were detected via ELISA. Days denote number of days after T cell injection. The “+” indicates significance above mean titer of sera from uninjected CB17 mice. NT, not tested.

The abilities of Th1 and Th2 cells, with and without FasL, to elicit autoantibody production, promote autoreactive B cell survival, and trigger participation in the GC reaction in vivo are summarized in Table I. These studies were done using anti-chromatin B cells from both VH3H9 Tg and site-directed VH3H9 Tg mice because of differences documented in signaling between IgM/IgD and IgG receptors (56, 57) as well as disparities seen between randomly integrated transgenes and those that have been targeted to the JH locus (58). The only distinction we detected between the site-directed and the non-site-directed transgenes was that the former generated isotype-switched Abs in response to T cell help.

Although polarized CD4+ T cell populations are typically described by the production of Th1 vs Th2 cytokines, it has been documented that within these populations a wide range of cytokines can be produced (29, 59). In this study, this variability was most pronounced for the Th1 polarized cells and proved to be significant in terms of B cell fate. Although Th2 cells consistently induced high titers of anti-chromatin Abs in a CD40-dependent manner, the ability of Th1-deviated cells to help anti-chromatin B cells correlated with T cell IFN-γ production. IFN-γhigh Th1 cells induced anti-chromatin AFCs and GCs, but IFN-γlow Th1 cells promoted only GCs, and a much lower degree of anti-chromatin B cell recovery. Unlike the tightly clustered anti-chromatin AFCs observed in Th2 recipients, the AFCs found under IFN-γhigh Th1 conditions were more diffuse and localized at the border of the T cell area, as has been described for other autoimmune AFCs in lpr/lpr or gld/gld mice (4, 60, 61, 62). This phenotype is even more pronounced in recipients of Th1-gld/gld cells, which is consistent with the possibility that the T cells driving the autoimmune response in Fas/FasL-mutant mice are of the Th1 type (61, 62, 63, 64, 65). The significance of the distinct localization sites for AFCs has not been determined, but in one model, these extrafollicular cells were somatically mutated (60).

FasL plays an important role in influencing the outcome of T cell help for anti-chromatin B cells. Relative to their FasL+ counterparts, Th2-gld/gld cells induced higher titers of IgM and IgG2a isotype anti-chromatin Abs, and more frequent anti-chromatin B cell GCs. Strikingly, the inability of IFN-γlow Th1 cells to induce anti-chromatin Ab production appears dependent on FasL, as only FasL-deficient IFN-γlow Th1 cells supported significant anti-chromatin B cell survival and high titers of anti-chromatin Abs.

FasL may alter anti-chromatin B cell fate indirectly by limiting T cell help (32, 33, 35) or by direct B cell killing (20). T cell loss due to FasL expression, however, was not observed in our study. IL-4, which has been shown to protect B cells from Fas-mediated death (66), may be responsible for the similar recovery of anti-chromatin B cells in recipients of Th2 FasL-sufficient and -deficient cells (see Fig. 5). The finding that IFN-γhigh, but not IFN-γlow, Th1 cell help results in anti-chromatin Ab production and B cell survival, prompts us to consider that, like IL-4 (66), IFN-γ may impart resistance to Fas-mediated death. Consistent with this hypothesis, one report showed a synergistic effect on B cells if IFN-γ was combined with stimulatory CpG oligonucleotides (67), but the direct effect of IFN-γ on B cells remains controversial (68, 69, 70, 71, 72, 73). An alternate hypothesis is that the IFN-γlow Th1 cells have diverged to a distinct differentiation pathway, such that they provide unique helper functions compared with the IFN-γhigh Th1 cells. Because we have previously documented that undeviated Th cells can promote autoantibody production (16), the failure of the IFN-γlow Th1 cells to induce autoantibodies is not likely to be a consequence of them being less differentiated.

It has been postulated that Fas-mediated killing is involved in B cell negative selection within GCs (74, 75, 76, 77). Our experiments provide no evidence that B cell death via FasL borne on Th cells curtails anti-chromatin GCs: Th1 cells (both IFN-γhigh and IFN-γlow) induce anti-chromatin GCs with or without FasL, and although Th2-gld/gld but not FasL-sufficient Th2 cells induce anti-chromatin GCs, a difference in B cell survival is not observed. Rather, we found a correlation between IFN-γ production by Th cells and B cell GC differentiation. In all cases where some degree of IFN-γ is produced (including Th2-gld/gld conditions), anti-chromatin B cells are found in GCs. Notably, older Fas/FasL-deficient mice have a predominance of IFN-γ+ T cells (78), and IFN-γ is critical for autoantibody production and autoimmune disease in MRL-lpr/lpr mice (79, 80). Studies to examine the direct effect of IFN-γ on anti-chromatin B cell proliferation, activation, and sensitivity to Fas-mediated death are under way.

Although we have documented that anti-chromatin B cells can form GCs, there is no evidence that long-lived AFCs are generated by the addition of T cell help (Table II). Other reports have described anti-DNA B cells in GCs in the absence of detectable serum Ab production (81, 82), suggesting a fail-safe mechanism that guards against memory formation and long-term autoantibody production. The observation documented here that only short-lived AFCs are generated in healthy mice by provision of T cell help suggests a means of curtailing the liability of autoreactive B cells. This conclusion is tempered, however, as another study, again using a transfer model but this time with nonautoreactive B cells, also failed to induce long-lived AFCs (26). This raises the possibility that their generation may have particular requirements that are not met under the transfer conditions used. Studies are underway to determine whether autoantibodies that arise naturally in autoimmune settings are derived from long-lived or short-lived AFCs. In the New Zealand Black/White and New Zealand M2410 models, the presence of long-lived AFCs has been documented, and interestingly, they have been shown to reside at unique sites (83, 84, 85). Clearly, more studies are warranted to better understand what governs B cell fate decisions in healthy vs autoimmune settings, the outcome of which may have important implications for B cell depletion strategies currently used to treat human autoimmune diseases (86, 87).

We thank A. Acosta and J. Faust of The Wistar Institute Flow Cytometry Facility; S. Alexander and A. Pagán for excellent technical assistance; and R. Noelle, M. Weigert, G. Trinchieri, and M. Monestier for reagents.

The authors have no financial conflict of interest.

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

Funding has been provided by the National Institutes of Health (AI32137, AR47913, and 2T32AI007518) and the Commonwealth Universal Research Enhancement Program, PA Department of Health.

3

Abbreviations used in this paper: Tg, transgenic; HA, hemaglutinin; FasL, Fas ligand; AFC, Ab-forming cell; AP, alkaline phosphatase; PNA, peanut agglutinin; GC, germinal center; EFF, extrafollicular foci.

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