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The Journal of Immunology, 2000, 165: 4095-4104.
Copyright © 2000 by The American Association of Immunologists

CD4+ T Lymphocytes with Constitutive CD40 Ligand in Preautoimmune (NZB x NZW)F1 Lupus-Prone Mice: Phenotype and Possible Role in Autoreactivity1

Helene Lettesjö, Gary P. Burd and Rizgar A. Mageed2

The Kennedy Institute of Rheumatology, Hammersmith, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lupus disease is marked by B lymphocyte hyperactivity and the production of Abs to dsDNA. The production of these anti-dsDNA Abs is T lymphocyte dependent. However, it is not clear how CD4+ T lymphocytes provide help for B lymphocytes to produce IgG anti-dsDNA Abs. One possible mechanism is suggested by studies showing that human patients with systemic lupus erythematosus and lupus mice have increased numbers of CD40 ligand (CD40L)+ T and B lymphocytes. The results described in this study reveal that young, clinically healthy lupus-prone New Zealand Black x New Zealand White F1 (BWF1) mice have naive CD4+ T cells with preformed CD40L. These cells contribute to a brisk response to immunization and to the production of anti-dsDNA Abs. In vitro experiments revealed that CD4+ T cells with preformed CD40L could, upon stimulation, provide antiapoptotic signals for B cells but could not induce proliferation or reduce activation threshold. These results suggest that the direct target cells for the effect of T cells with preformed CD40L in lupus may not be B lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE)3 is a prototype autoimmune rheumatic disease manifested by many immunological abnormalities. The most evident of these abnormalities is the production of potentially pathogenic Abs to nuclear Ags, including Abs to dsDNA (1). Despite tangible improvement in the overall outcome of diagnosis and treatment, SLE continues to cause considerable morbidity and mortality. Therefore, there is a continuing need to improve understanding of lupus pathogenesis and seek new therapeutic targets.

The New Zealand mouse model of lupus, the BWF1 mouse, which results from crossing the New Zealand Black (NZB) mouse with the New Zealand White (NZW) mouse, inherits B lymphocyte hyperactivity from the NZB parent and develops Abs to dsDNA accompanied by severe, rapidly progressive nephritis with onset at ~5 mo of age (2). The model is a good representative of human SLE because female BWF1 mice manifest similar autoantibody profiles, and the disease develops in the absence of any known disease-accelerating gene (3). Production of anti-dsDNA Abs in this mouse, as in human SLE, is thought to be T cell-dependent as suggested by the IgG isotype, somatic mutations in the Ab genes, and by experiments in which depletion of T cells suppressed Ab production and ameliorated disease (4, 5). However, although several potential mechanisms have been proposed to explain cognate T-B cell interaction in this setting (6, 7), it remains unclear how CD4+ T cells, which recognize peptide-MHC class II complexes provide help for anti-dsDNA Ab-producing B cells in lupus. An alternative possibility to cognate T-B cell interaction leading to the production of anti-dsDNA Ab is intrinsic lymphocyte defects resulting in aberrant responses including anti-DNA Ab production. In this respect there is evidence that mutations in the Fas-Fas ligand genes in the MRL-lpr and gld mice, polymorphism in genes encoding elements of B cell receptor (BCR) signaling, and noncognate up-regulation of accessory molecules could lead to anti-dsDNA Ab production (8, 9, 10). Relevant to the specific immunological abnormalities in BWF1 mice and human SLE patients is the finding of increased numbers of T and B lymphocytes with functional CD40 ligand (CD40L) in secondary lymphoid tissues and blood of lupus mice and SLE patients, respectively (11, 12). Thus, engagement of CD40 by CD40L on B cells can reduce the threshold of B cell activation, which may partly explain spontaneous Ig production and the tendency to switch Ig isotype in the absence of T lymphocyte help (13). Furthermore, engagement of CD40 on B cells by CD40L up-regulates Bcl-xL and Bcl-2 expression, which can override the propensity of germinal center B cells to die by apoptosis, or block tolerance induction in naive B cells (14, 15). Direct evidence for the important role of the CD40-CD40L pathway in lupus disease comes from studies in which mouse models of lupus have been successfully treated with blocking anti-CD40L Ab (11, 16). However, fundamental issues remain unresolved. For example, it is not clear whether the finding of CD40L+ lymphocytes is due to intrinsic defects or to ongoing lymphocyte activation by self-Ags and the accumulation of memory cells. Furthermore, the specificity of the findings to lupus autoimmunity and role in disease mechanisms remain speculative (17, 18).

Activation of T lymphocytes by Ag-MHC complexes is the primary pathway leading to CD40L up-regulation, and the process is tightly regulated to prevent bystander activation of lymphocytes (19). Regulation of CD40L expression involves transcriptional and posttranscriptional pathways, mRNA stability, and shedding of membrane CD40L protein by proteases (20, 21, 22). However, it is known that memory T lymphocytes retain preformed cytoplasmic CD40L (23). In mice, these cells congregate in the T cell areas of lymphoid organs in readiness for secondary responses to Ags (24). Evidence from transgenic mice with preformed CD40L shows that preformed CD40L helps in mounting brisk responses to immunogens (25). In humans, CD45RO+ T memory cells with cytoplasmic CD40L gather in the light zone of germinal centers and rapidly up-regulate CD40L upon stimulation (24). Subsequent studies showed that these T cells have the capacity to rescue newly generated centrocytes from apoptosis (26).

In this study, we have investigated whether CD40L is abnormally regulated in lupus-prone BWF1 mice. Furthermore, experiments were conducted to determine whether, and how, CD4+ T cells with preformed CD40L could contribute to some immunological features of lupus autoimmunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

NZB, NZW, BWF1, and CBA/HT6 mice were bred and maintained at the breeding facility of the Kennedy Institute of Rheumatology. Mice from these in-house bred groups were used for immunohistology, immunization, and flow cytometry. For some of the later flow cytometric analyses and functional studies young (3–4 wk old) CBA mice were purchased from Olac (Bicester, U.K.) and left to acclimatize at the animal facility for 2 wk before use.

Antibodies

Monoclonal hamster Abs to mouse CD40L (clone MR1) and dinitrophenol (clone UC8-1B9, used as control) were obtained from the American Type Culture Collection (Manassas, VA). PE-conjugated rat anti-mouse CD44, FITC-conjugated rat anti-CD45RB, FITC rat anti-CD62L, FITC hamster anti-CD69, FITC hamster IgG antikeyhole limpet hemocyanin (KLH; negative control for anti-CD69 staining), PE hamster IgG antitrinitrophenol control Ig, and CyChrome-streptavidin were obtained from PharMingen (San Diego, CA). FITC rat anti-CD25, FITC rat anti-CD4, PE-Extravidin, and FITC-Extravidin were obtained from Sigma (Dorset, U.K.). PE rat anti-B220 was obtained from Caltag (Burlingame, CA); PE rat anti-CD80 (B7-1) and PE rat anti-CD86 (B7-2) were obtained from Cedarlane (Ontario, Canada); FITC rat IgG2a, PE rat IgG2a, FITC mouse IgG1 (all controls), and FITC anti-CD44 were obtained from Serotec (Oxford, U.K.). To block nonspecific binding, 200 µg/ml purified hamster IgG (Cappel; Organon Teknika, West Chester, PA) or rat IgG (Serotec) were incubated with the tissues and cells before addition of the specific Abs.

Immunohistology

Spleens from naive mice or mice immunized with phosphorylcholine (PC)-conjugated BSA were frozen in liquid nitrogen in optimum cutting tissue. Six-micron sections were affixed onto gelatin-coated slides and fixed in acetone at 4°C. Twenty percent goat serum in PBS was incubated on the sections for 1 h at room temperature to prevent nonspecific binding of biotinylated anti-hamster IgG. Purified monoclonal anti-mouse CD40L Ab (clone MR1) was incubated on each section overnight at 4°C. Negative controls included sections stained with polyclonal hamster IgG (Cappel). To confirm specificity of staining with the MR1 Ab, sections were preincubated with soluble mouse CD40-human IgG1 Fc recombinant protein (27) (provided by Dr. David Gray, Edinburgh University, Edinburgh, U.K.) to block CD40L before adding MR1 for overnight incubation. The sections were washed with PBS and incubated with biotinylated goat anti-hamster IgG mixture (PharMingen) in PBS/0.02% goat serum. The slides were developed with avidin-biotin HRP complex (Vector Laboratories, Burlingame, CA) followed by 3,3'-diaminobenzidine tetrahydrochloride solution (Sigma).

Membrane and cytoplasmic staining by flow cytometry

Half a million isolated CD4+ T cells (see below) were stained in 50 µl PBS containing 1% BSA and 0.02% sodium azide (PBS/BSA/azide) for 30 min at 4°C with primary Abs for membrane staining and at room temperature in PBS/BSA with 1% saponin (Sigma) for cytoplasmic staining. Extravidin or streptavidin conjugates were added to cells stained with biotin-conjugated Abs and incubated for 20 min at 4°C. Cells were fixed for 20 min at 4°C with 2% formaldehyde before cytoplasmic staining and after membrane staining. For cell cycle analyses, a final concentration of 5 µg/ml propidium iodide (Sigma) and 10 µg/ml RNase A (Ambion, Austin, TX) were added to intracellularly stained cells. All FACS samples were analyzed on a FACScan (Becton Dickinson) and analyzed using Win MDI 2.7 software.

Isolation and activation of CD4+ T lymphocytes

CD4+ T cells were isolated from single-cell suspension of splenocytes using magnetic beads coated with anti-mouse CD4 (Dynal, Oslo, Norway). In brief, CD4+ T cells were attached to anti-CD4-coated beads by incubation for 30 min at 4°C, and bound cells were released with anti-CD4 detachabeads. For separation of small and large T cells, a discontinuous Percoll gradient (Pharmacia, Uppsala, Sweden) with 50, 60, 65, 72, and 100% Percoll was used. Small cells were collected at the interface between 65 and 72% and large cells between 50 and 60%. For studying the kinetics of CD40L up-regulation, CD4+ T lymphocytes were cultured at 106 cells/well in 1 ml medium in a 24-well plate and stimulated for various time periods with 10 ng/ml PMA together with 1 µg/ml ionomycin (Sigma). To study the kinetics of up-regulation of preformed cytoplasmic CD40L, the cells were cultured in the presence of 20 µg/ml cycloheximide (CHX) (Sigma) together with PMA/ionomycin. The effect of CHX on de novo protein synthesis was studied by monitoring the expression of CD25 on PMA/ionomycin-stimulated CD4+ T cells. A range of CHX concentrations, 0.5, 1, 2.5, 5, and 10 µg/ml was used for treating freshly isolated CD4+ T cells together with PMA/ionomycin. The dose of 10 µg/ml of CHX produced >95% inhibition of CD25 expression by T cells activated with PMA/ionomycin. On this basis, the experiments involving CHX were all conducted using 20 µg/ml CHX, and the cells were maintained with CHX during the entire culture time unless otherwise indicated. For phenotype characterization, the cells were stimulated for 15 min, 30 min, 1 h, or 2 h before determining CD40L membrane expression on memory and naive triple-stained cells. For coculture of T cells with B cells, T cells were stimulated for 2 h at 37°C with 10 ng/ml phorbol-dibutyrate (PDBu) (Sigma) and 1 µg/ml ionomycin, with or without CHX, and the cells were washed three times to remove all traces of the PDBu/ionomycin. One-third of the cells were left unstimulated. T cells were then fixed with 1 ml 2% formaldehyde for 10 min at 4°C before coculturing with the B cells.

Isolation of B cells

B cells were isolated after RBC depletion. T lymphocytes were depleted using a mixture of anti-Thy1 (clone AT-83 A), anti-CD4 (clone YTA 3.1.2). and anti-CD8 (clone 53.6.72-14), together with rabbit complement (Cedarlane). Macrophages were removed by plastic adherence and by the use of 5 mM L-leucine methyl ester (Sigma). Residual T cells were removed by anti-Thy1-coated magnetic beads (Dynal). Dead cells and debris were removed using a Lymphocyte M gradient (Cedarlane).

Functional studies

Cultures described in this section were set up in RPMI 1640 with L-glutamine (Whittaker Bioproducts, Verviers, Belgium), supplemented with 10% FCS, penicillin, streptomycin, and 5 x 10-5 M 2-ME (Life Technologies, Paisley, U.K.). B cell proliferation. To determine whether cytoplasmic CD40L+ (cCD40L+) CD4+ T cells could induce B cell proliferation, 5 x 104, 1 x 105, or 2 x 105 B cells from naive or PC-BSA-immunized BWF1 mice were mixed with PDBu/ionomycin-prestimulated (2 h; with or without CHX) T cells from naive BWF1 mice, to give ratios of 5:1, 2:1, or 1:1 B:T cells for each B cell population (i.e., 5 x 104 B cells with 1 x 104, 2.5 x 104, or 5 x 104 T cells; 1 x 105 B cells with 2 x 104, 5 x 104, or 1 x 105 T cells, etc.). All T cells were fixed to maintain the same level of CD40L on the membrane throughout the culture period. In all experiments, the cells were cocultured for 3 days in 200 µl medium. Parallel experiments were conducted under the same conditions but with mouse IL-4 in the medium. In all experiments where positive results were obtained, the role of the CD40L was confirmed by blocking with 10 µg/ml of purified MR1 Ab, or the isotype control. To study the effect of cCD40L+CD4+ T cells on the threshold of B cell stimulation through the BCR, dose-response experiments with a range of three anti-IgM Ab concentrations were conducted. After 24-h coculture of 2 x 105 fixed CD4+ T cells prestimulated with PDBu/ionomycin for 2 h with or without CHX, with 2 x 105 purified B cells, the B cells were then stimulated with 0.025, 0.25, or 2.5 µg/ml anti-IgM. For proliferation, the cells were pulsed with 0.5 µCi [3H]thymidine/well during the final 18 h of incubation. B cell differentiation. Half a million PDBu/ionomycin-stimulated and fixed CD4+ T lymphocytes cells with or without CHX were cocultured with 0.5 x 106 B cells from naive or immunized BWF1 mice in the absence or presence of 10 ng/ml mouse IL-4 and 10 ng/ml IL-10 (Schering-Plough, Madison, NJ). Supernatants were collected after 7 days and tested for Igs and Abs to DNA and to nucleosome by ELISA. B and T lymphocyte aggregation. One million PDBu/ionomycin-stimulated CD4+ T lymphocytes, with or without CHX, from naive 6-wk-old BWF1 mice and 106 B lymphocytes from PC-BSA-immunized mice (day 7), were cocultured for 20 h at 37°C. Two hundred thousand cells were washed with PBS/2% FCS, stained with PE-anti B220 and FITC-anti CD4 for 30 min at 4°C, and analyzed on a FACScan. B-T lymphocyte aggregation was determined by the detection of double-positive B and T cells (26). B cell survival. Half a million PDBu/ionomycin-treated fixed CD4+ T lymphocytes cells, with or without CHX, from naive 6 wk BWF1 mice and 0.5 x 106 B lymphocytes from either naive or immunized BWF1 mice (day 7) were cocultured for 4 h at 37°C in 0.5 ml medium. Control cultures were set up with 10 µg/ml MR1 Ab or isotype control. Two hundred thousand cells/FACS tube were stained with PE-anti B220 for 20 min at 4°C and washed with PBS. One hundred microliters of PBS were added together with 7-amino actinomycin D (7-AAD; Sigma) at a final concentration of 5 µg/ml. After a 15-min incubation at room temperature, 400 µl PBS was added and the samples were immediately analyzed on a FACScan.

Immunization

Primary immunization of BWF1 and CBA mice was conducted by i.p. injection of 100 µl (containing 250 µg) PC-BSA in CFA (1:1 mix; Difco, Detroit, MI). Secondary immunization was conducted in IFA (1:1 mix) 7 days after the primary immunization. PC was conjugated to BSA using a protocol adapted from that published by Pery et al. (28). The effect of immunization with PC-BSA on the progression of spontaneous disease in BWF1 mice was assessed in experiments in which mice were either immunized with PC-BSA as above, or with PBS emulsified in CFA and IFA. In these experiments, two groups of 6- to 8-wk-old female BWF1 mice were used. The first group of 14 mice was immunized with PC-BSA. The second group of 10 mice received 100 µl of PBS in CFA and IFA. The effect of immunization on disease progression in both groups of mice was analyzed by determining survival, proteinuria, and IgG anti-dsDNA Abs. Proteinuria was measured using Albym-Test dipsticks (Boehringer-Mannheim, Mannheim, Germany), where a result of 0 indicates no protein in the urine, +1 is <30 g/L, +2 is <100 g/L, and +3 is >100 g/L. For the purpose of this investigation, a mouse with a proteinuria level of >100 g/L was deemed "proteinuria positive."

To study the effect of blocking the CD40-CD40L pathway on the outcome of the immunization, four injections each of 500 µg purified sterile MR1 Ab, or the isotype control (anti-dinitrophenol Ab clone UC8-1B9), were given at 2-day intervals; 2 days before immunization, on the day of immunization, and 2 and 4 days after the immunization.

ELISA for the detection of Abs to BSA and PC in mouse serum

Abs to BSA and PC were detected using ELISA plates coated with 10 µg/ml BSA, 20 µg/ml of PC-conjugated to KLH, or 20 µg/ml KLH (as control) in PBS. Sera were added at a dilution of 1:50 and titrated at 1:2. Bound IgG or IgM Abs were revealed with alkaline phosphatase-conjugated anti-mouse {gamma}- or µ-chains (Sigma). OD values were determined at 405 nm, and results were expressed as units. These units were determined by taking a ratio between the OD value for the test sample to that obtained for the positive control at the same dilution (pooled sera from normal mice immunized with PC-BSA), which was considered to represent 100% binding value.

ELISA for total IgM and IgG and Abs to ssDNA, dsDNA, and nucleosomes

Total IgG or IgM were measured by ELISA using plates coated with 10 µg/ml of goat F(ab')2 anti-mouse {gamma}- or µ-chains (Caltag), respectively. For the detection of Abs to ssDNA, dsDNA, and nucleosomes, the plates were coated with methylated BSA (mBSA; Calbiochem, Nottingham, U.K.) (6, 29). About 100–200 bp long dsDNA fragments of calf thymus DNA (Sigma) were added to mBSA-coated wells at 50 µg/ml in PBS. For detecting Abs to ssDNA, dsDNA was boiled and chilled on ice before adding to the mBSA-coated wells. For detecting Abs to nucleosome, dsDNA-coated plates were coated with 20 µg/ml total histone (Sigma). Supernatants (or 1/50 dilutions of serum) were added to the plates and titrated at 1:2. Bound IgM and IgG were revealed with HRP-conjugated goat anti-mouse {gamma}- or µ-chains (Caltag). Positive and negative controls were included to confirm specificity. The results were expressed as ELISA units by taking a ratio between the OD value for each sample relative to the OD value for 1:100 dilution of a pooled serum from old anti-ssDNA, -dsDNA, and -nucleosome Ab-positive BWF1 mice.

Statistical analysis

Differences between BWF1 and CBA mice in the number of T cells with cCD40L, proliferation, and ELISA results were determined using Student’s t test. Differences between BWF1 mice immunized with PC-BSA and those receiving PBS in CFA/IFA in the number of surviving mice and those developing proteinuria were analyzed using the {chi}2 test. Differences were considered statistically significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD40L+ cells are detectable in the spleen of young, clinically healthy BWF1 mice

Immunohistological analyses showed that spleen sections from young, clinically healthy BWF1 mice had significantly higher numbers of CD40L+ cells than CBA mice (p < 0.001; Table IGo). The mean number of CD40L+ cells around terminal arterioles was 20 times higher in the BWF1 than in the CBA mice (BWF1 = 4.5 ± 0.8; CBA = 0.2 ± 0.1). CD40L+ cells were generally found around terminal arterioles (TA) and in the outer periarteriolar lymphoid sheath (PALS) areas (Fig. 1Go). To confirm the immunohistology data, membrane and cytoplasmic CD40L expression were studied by flow cytometry using freshly isolated CD4+ T cells. Fig. 2Go shows that BWF1 mice had higher numbers of membrane CD40L+ (mCD40L+) CD4+ T cells compared with CBA mice. The difference in the number of cytoplasmic CD40L+ (cCD40L+) T cells between BWF1 and CBA mice was more pronounced than mCD40L. The results implied that most of the CD40L detected by immunohistology in BWF1 spleens was probably cytoplasmic. The higher number of cCD40L+ cells detected by flow cytometry compared with histology was partly due to the superior performance of the anti-CD40L mAb (MR1) in flow cytometry and partly due to the use of saponin to stain isolated (>95% pure) T cells in flow cytometry compared with histology in which the T cells constitute a fraction of the cells in the section.


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  		Table I. Number of CD40L-positive cells in different splenic compartments of PC-BSA-immunized BWF1 and CBA mice1

 


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  		FIGURE 1. Immunostaining for CD40L in spleen sections from BWF1 mice. Frozen spleen sections from young 4- to 6-wk-old clinically healthy nonimmunized BWF1 mice were stained with monoclonal MR1 hamster anti-mouse CD40L (A). To confirm specificity, frozen sections from the same spleens were preincubated with soluble mouse CD40-human IgG1 Fc recombinant protein to block CD40L before adding MR1 and stained (B). The sections are at x40 magnification.

 


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  		FIGURE 2. Flow cytometry for CD40L expression in isolated CD4+ T cells from young BWF1 and CBA mice. FACS analysis was performed after positive selection of CD4+ T cells using anti-CD4 mAb-coated magnetic beads. Cells were stained for membrane and intracellular CD40L. The percentage of CD40L+CD4+ T cells indicates the percentage of cells staining with MR1 after subtracting the number of cells stained with the control hamster mAb (clone UC8-1B9). A representative experiment of four is shown.

 
To confirm that CD4+ T cells from BWF1 mice contained preformed cCD40L, isolated CD4+ T cells were stimulated with PMA and ionomycin with or without CHX. Preliminary experiments to assess the effect of CHX treatment on de novo protein synthesis showed that treatment of T cells with 10 µg/ml CHX prevented CD25 up-regulation following PMA/ionomycin stimulation by >95%. In addition, no up-regulation of mCD40L was observed in activated T cells from nonimmunized CBA mice (data not shown). The effect of CHX treatment on CD40L up-regulation was similar to those previously observed for human naive CD45RA+ tonsillar T cells (23). Fig. 3Go, A–C show that the number of mCD40L-expressing cells was greatly enhanced after 2 h of PMA/ionomycin stimulation. When blocking CD40L synthesis with CHX, the number of mCD40L+ cells was still enhanced on T cells from BWF1 mice compared with unstimulated cells, confirming that BWF1 CD4+ T cells had preformed cCD40L that could be up-regulated on the cell surface after stimulation.



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  		FIGURE 3. CD40L is held intracellularly in CD4+ T cells from BWF1 mice. Membrane CD40L expression by CD4+ T cells before (A), or after 2 h of stimulation with PMA/ionomycin without (B), or with (C) CHX. Percentage of CD40L+ T cells indicates the percentage of cells stained with biotinylated MR1 after subtracting the percentage of cells stained with the biotinylated control hamster IgG. D, MFI of membrane CD40L expression in isolated CD4+ T cells after PMA/ionomycin activation with or without CHX for 15 min, 30 min, 1 h, and 2 h. To determine the kinetics of CD40L expression by CD4 T cells with a naive or memory phenotype, the cells were triple-stained with Abs to CD44, CD45RB, and biotinylated MR1. CD40L expression on cells gated for CD44+CD45RB- (memory phenotype) and on CD44-CD45RB+ (naive phenotype). Biotinylated hamster IgG was used to subtract nonspecific staining.

 
To determine the kinetics of preformed cCD40L transit to the membrane, isolated CD4+ T cells were stimulated with PMA/ionomycin together with or without CHX. After 15 min, 30 min, 1 h, and 2 h, the cells were stained for membrane CD44, CD45RB, and CD40L to compare the kinetics of mCD40L expression in T cells of the naive and memory phenotype. The mean fluorescence intensity (MFI) of mCD40L expression on T cells of the memory and naive phenotype at different time points following stimulation is shown in Fig. 3GoD. After 15 min (0.25 h) of activation, both T cells with naive and memory phenotypes treated with CHX up-regulated mCD40L. However, more CHX-treated CD4+ T cells of memory phenotype expressed high mCD40L compared with naive cells.

Kinetics of CD40L up-regulation in vivo is different in BWF1 mice compared with CBA mice

The kinetics of CD40L expression in vivo was studied following immunization (Table IGo). After primary immunization the highest number of CD40L+ cells in BWF1 mice was detected at 18 h around TA and in the outer PALS areas. Thereafter, the number of CD40L+ cells dropped rapidly, and by 24 h few CD40L+ cells were visible around TA or the PALS areas of BWF1 mice; instead, some CD40L+ cells were visible inside B cell follicles. In contrast, in CBA mice the peak number of visible CD40L+ cells was reached at 24 h after immunization. After secondary immunization, greater and sustained numbers of CD40L+ cells were seen in both strains.

Most cD40L+CD4+ T lymphocytes in young preautoimmune BWF1 mice display naive phenotype and are nondividing cells

To further determine the proportion and phenotype of cCD40L+ cells in young BWF1 mice, isolated CD4+ T cells were double-stained with FITC-conjugated Abs to membrane CD44, CD45RB, CD62L, CD69, or CD25 together with biotinylated anti-CD40L for cytoplasmic staining. Fig. 4Go shows that about 0.4 and 0.3% of CD4+ T cells had cCD40L and expressed the early activation markers CD69 and CD25. The majority of the cCD40L+ T cells (>80%) were of a naive CD44lowCD62LhighCD45RBhigh phenotype, whereas ~10% were of the CD44highCD62lowCD45RBlow memory phenotype. However, the brightest stained cCD40L+ cells were of the memory phenotype. Furthermore, the experiments also revealed that only ~6% of the cCD40L+CD4+T cells were in the dividing phase (Table IIGo). Previous studies have shown that the spleen contains a small number of naturally activated large T cells (30), which may contain preformed cCD40L. To study whether CD40L+CD4+ cells in BWF1 mice represent such a population, CD4+ T cells from BWF1 mice were separated on a Percoll gradient. Cells with cCD40L were distributed equally between small and large cells (data not shown). These experiments imply that whereas the vast majority of cCD40L+CD4+ T cells in BWF1 mice were of the naive phenotype, those with high levels of cCD40L were of the memory phenotype.



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  		FIGURE 4. Flow cytometry of surface phenotype expression of cCD40L+CD4+ T cells from BWF1 mice. Most cCD40L+CD4+ T cells from BWF1 mice expressed a naive phenotype. FACS analysis of positively selected CD4+ T cells was conducted using Abs to CD44, CD62L, CD45RB, CD69, and CD25. The stained cells were then permeabilized and stained for intracellular CD40L using biotinylated MR1. A representative experiment of five is presented.

 

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  		Table II. Cell cycle analysis of total CD4+ T cells and cytoplasmic CD40L+ CD4+ T lymphocytes in BWF1 mice1

 
cCD40L+CD4+ T cells relate to a brisk response to immunization and to induced anti-DNA autoantibody production in vivo

To investigate possible functional significance of preformed cCD40L to immune responses to exogenous Ags and to the production of anti-dsDNA Ab, young BWF1 and CBA mice were immunized with PC-BSA. This approach was chosen based on studies in which mice immunized with PC conjugated to KLH, or to BSA, produced Abs with dual specificity for PC and DNA (31). Table IIIGo shows that immunization of BWF1 mice resulted in a brisk T-dependent humoral response to BSA. Thus detectable levels of IgM were found by day 4, and by day 7 significant levels of both IgM and IgG Abs were found in BWF1 mice. This was in contrast to CBA mice in which detectable IgM levels were found by day 7–8. In addition, IgG anti-dsDNA Abs were detectable in the serum of BWF1 mice, but not CBA mice, by day 10. To confirm that preformed cCD40L+ T cells had a role in the brisk humoral response and anti-dsDNA Ab production, CD40L was blocked with 500 µg purified sterile MR1 2 days before immunization and on days 0, 2, and 4 after immunization. This treatment protocol resulted in suppression of the immune response to PC-BSA (not shown) and to a complete blockade of IgG anti-DNA Ab production. These data, although not discriminating between the role played by preformed cCD40L and de novo synthesized CD40L, nevertheless suggest that the CD40-CD40L pathway plays a major role in the production of dual PC and DNA reactive Abs in PC-BSA immunized BWF1 mice.


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  		Table III. Anti-BSA Ab isotype and IgG anti-dsDNA responses in PC-BSA-immunized BWF1 and CBA mice1

 
The possible biological significance of up-regulated CD40L to lupus disease was further assessed by long-term monitoring of anti-dsDNA autoantibody levels, proteinuria, and survival of mice immunized with PC-BSA compared with mice injected with PBS in CFA. Two groups of 14 and 10 young clinically healthy female BWF1 mice were immunized with PC-BSA or PBS, respectively, and monitored for serological and clinical evidence of lupus. Fig. 5Go shows that mice immunized with PC-BSA developed higher levels of anti-dsDNA Abs and proteinuria sooner and died earlier than mice injected with PBS in CFA.



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  		FIGURE 5. Immunization of young BWF1 mice with PC-BSA accelerates the development of IgG anti-dsDNA Ab, proteinuria, and death. A, Mean and SEM of IgG anti-dsDNA Abs detected by ELISA in 14 BWF1 mice immunized with PC-BSA ({blacksquare}) and 10 BWF1 mice injected with PBS emulsified in CFA ({square}) at 4–6 wk of age. ELISA units are arbitrary determined by taking a ratio between the OD value obtained for the test sample to that obtained for the positive control sample at the same dilution that was considered to represent 100% of the binding value to dsDNA. B, Percentage of living BWF1 mice from both groups with >100 g/L of protein in their urine after immunization with PC-BSA ({blacktriangleup}) or injection with PBS in CFA ({blacksquare}). C, Percentage of surviving BWF1 mice after immunization with PC-BSA ({blacktriangleup}) or injection with BSA in CFA ({blacksquare}). Statistical differences between the two BWF1 groups in IgG anti-dsDNA levels were determined using the nonpaired t test, whereas differences in numbers of mice with proteinuria and survival were determined using the {chi}2 test. *, p < 0.05; **, p < 0.01.

 
Functional role of cCD40L+CD4+ T cells in T-B cell cocultures in vitro

B cell proliferation. The results of proliferation studies showed that membrane CD40Lhigh T cells, which represent stimulated T cells with de novo synthesized CD40L in the absence of CHX, but not CHX-treated cCD40Llow T cells from nonimmunized mice, were capable of inducing B cells from immunized mice to proliferate (Fig. 6Go). However, neither T cell population could induce the proliferation of B cells from naive mice. Suboptimal doses of anti-IgM (<2.5 µg/ml) added 24 h after coculture of resting B cells with stimulated T cells enhanced proliferation only with CD40Lhigh but not CD40Llow cells (data not shown), suggesting that naive CD4+ T cells with preformed CD40L could not reduce the threshold of suboptimally activated B cells without de novo CD40L synthesis (32).



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  		FIGURE 6. Induction of B cell proliferation by cCD40L+CD4+ T cells. Proliferation of B cells from PC-BSA-immunized (A), or naive (B) mice after 3 days of coculture with PDBu/ionomycin-activated CD4+ T cells (2 h) from young naive BWF1 mice in the absence of CHX (mCD40Lhigh), or with CHX (mCD40Llow). [3H]Thymidine was added during the last 18 h of the cocultures. 3H incorporation is shown as mean cpm ± SEM of triplicate cultures. Purified MR1, or the control Ab, was added to parallel cocultures to determine the role of CD40L. One representative experiment of two is shown.

 
B cell differentiation. Upon coculture with stimulated T cells with PDBu/ionomycin (mCD40Lhigh), B cells from immunized mice produced IgM and IgG Abs. Some of the IgM Abs reacted with dsDNA, ssDNA, and nucleosomes (data not shown). Blocking CD40L at the beginning of the culture period reduced IgM and IgG Ab levels. B cells cocultured with PDBu/ionomycin/CHX-stimulated T cells (mCD40Llow) had a significant increase (p < 0.05) in IgM compared with B cells cocultured with unstimulated CD4+ T cells. However, no IgG was detected. Addition of IL-4 and/or IL-10 did not enhance Ab production.

T-B cell aggregation. Very few B-T cell aggregates were detected after 20 h of coculture in all cultures regardless of the manner of T cell stimulation before coculture (data not shown). CD86 expression was enhanced by mCD40Lhigh but not by mCD40Llow cells. However, blockade of CD40L with MR1 Ab at the start of the culture was unable to block up-regulation of CD86 expression. No expression of CD80 was observed.

B cell survival. Coculture experiments were set up with unstimulated or PDBu/ionomycin-stimulated CD4+ T cells, with or without CHX, together with B cells from naive or immunized BWF1 mice to determine whether cCD40L+CD4+ T cells could rescue syngeneic B cells from apoptosis. After 4 h of coculture, survival of B cells was determined by estimating the number of B220+7-AAD- (living) cells by flow cytometry. Fig. 7Go shows that the number of surviving B cells from immunized mice was enhanced in cultures with mCD40Llow T cells from nonimmunized mice compared with cultures with unstimulated T cells. The survival of B cells cocultured with mCD40Lhigh T cells was more pronounced. Addition of MR1 Ab almost completely blocked B cell survival in cocultures with mCD40Lhigh cells, but only partially blocked survival with mCD40Llow T cells. Coculturing mCD40Lhigh T cells with B cells from naive mice had no effect on B cell survival after 4 h, but after 20 h of coculture an increased number of surviving B cells was found. In contrast, cCD40L+CD4+ T cells from nonimmunized mice were unable to increase B cell survival from naive mice at any time point (data not shown).



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  		FIGURE 7. cCD40L+CD4+ T cells can provide antiapoptotic signals to B cells. Survival of B cells from PC-BSA-immunized BWF1 mice cocultured with PDBu/ionomycin-stimulated CD4+ T cells (for 2 h) from young naive BWF1 mice in the absence of CHX (mCD40Lhigh) or with CHX (mCD40Llow). The stimulated T cells were fixed to maintain surface CD40L levels before coculture with B cells for 4 h. Cells were stained with 7-AAD and Abs to B220. 7-AAD-B220+ cells were counted as surviving B cells. The increase in B cell survival was determined by calculating the increase in the percentage of 7-AAD-B220+ cells recovered from cultures with stimulated T cells compared with the percentage of 7-AAD-B220+ cells recovered from cultures with unstimulated T cells. mAb MR1, or isotype control, was added to parallel cultures under identical conditions to determine the role played by CD40L in B lymphocyte survival. One representative experiment of two is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A prominent feature of lupus is autoimmunity to nuclear Ags, including the production of Abs to dsDNA. Production of these Abs is T cell-dependent as evident by the IgG isotype, somatic mutations in the variable region genes, and experiments in which Abs to Thy-1 or CD4 suppressed anti-dsDNA Ab production and ameliorated disease (5). However, it remains unclear how CD4+ T cells, which recognize peptide-MHC class II complexes, provide help for B cells to produce anti-dsDNA Abs. Suggested mechanisms include T cell recognition of DNA-associated histones, recognition of peptide fragments of IgG anti-dsDNA Abs, and T cell recognition of CD1, an invariant MHC class I-like molecule expressed on a subset of B cells (7, 33, 34).

An alternative, though not mutually exclusive, mechanism to T cell-driven response to nuclear Ags is that anti-dsDNA production results from hyperresponsiveness of B cells. Evidence in support of this mechanism is provided by studies in which B cells from BWF1 mice spontaneously produce Igs in vitro and switch isotype in the absence of T cell help when passively transferred to immune-deficient mice (13). In this study, Reininger and colleagues showed that transfer of bone marrow pre-B cells from BWF1 mice led to autoantibody production and isotype switch in SCID and RAG-/- mice in the absence of T cell help. Furthermore, it has long been known that induction of tolerance to protein Ags in the B cell compartment is difficult to achieve in adult BWF1 mice (35). More recently, a B lymphocyte hyperactivity syndrome resembling lupus has been described in mice lacking the Src family kinase Lyn, which is an inhibitory component of BCR signaling. These mice spontaneously produced anti-dsDNA Abs (9). A number of other studies have suggested that lymphocytes in lupus disease exhibit abnormality in regulating the expression of accessory molecules. Thus, studies have shown increased numbers of CD40L+ T cells in mice and human patients with lupus in the apparent absence of stimulation through BCR/TCR and enhanced ability to express costimulatory molecules by B cells in response to a variety of stimuli (10, 11, 12, 36). However, it has not been possible to establish whether the presence of CD40L+ T cells in lupus-prone mice and human patients is due to stimulation by autoantigen or due to intrinsic lymphocyte defects. Another possibility that may further confuse efforts to identify the primary cause of CD40L+ up-regulation is that dysregulation of the cytokine network could lead to noncognate expression of CD40L. For example, activation of the innate immune system leads to IL-12 production by dendritic cells and by macrophages, which in turn leads to CD40L up-regulation through IFN-{gamma} production (37, 38).

The experiments described in this study were conducted with two aims in mind; first, to determine whether CD40L up-regulation in young preautoimmune BWF1 mice is an intrinsic feature or due to activation, and second, to explore the possible role of cCD40L+ T cells in autoimmunity in the BWF1 mouse. The results show that T cells from naive clinically healthy BWF1 mice, in contrast to normal mice, have CD40L+ T cells in the T cell area of the spleen before the onset of clinical or serological signs of lupus autoimmunity.

Previous studies of CD40L expression in lupus have shown that although human SLE patients in remission had CD40L+CD4+ T cells in equal numbers to normal individuals, T cells from patients up-regulated higher levels of CD40L following mitogen stimulation in vitro. These studies implied that increased numbers of CD40L+CD4+ T cells in the circulation of SLE patients were due to intrinsic defects resulting in lowered threshold for CD40L expression (10). Although the experiments conducted in our study did not directly test this possibility, the results are, nevertheless, consistent with the notion that cells with cCD40L did not express a phenotype associated with activated T cells.

To explore the influence of cCD40L+ T cells on the immune system in BWF1 mice and in anti-dsDNA production, a new approach was used. This approach is based on immunizing young BWF1 (and normal CBA) mice with PC-BSA. This approach was used because identifying the contribution of cCD40L+ T cells to spontaneous anti-dsDNA Ab production in old BWF1 mice would have been difficult because of the advanced state of the autoimmune response. PC has a structure similar to the backbone of DNA, and previous studies have shown that anti-PC Abs acquire reactivity for dsDNA after undergoing a single nucleotide mutation in the heavy chain variable region gene (39). In addition, more recent studies have shown that immunization of young preautoimmune BWF1 mice with PC conjugated to a carrier protein (KLH or BSA) led to anti-dsDNA Ab production (31).

The results of immunization of BWF1 mice with PC-BSA showed that these mice had a brisk response to PC and to BSA and produced anti-dsDNA Abs. Furthermore, these mice had an accelerated proteinuria and death following immunization. As was expected, pretreatment of the immunized BWF1 mice with anti-CD40L slowed the response to PC and to BSA and suppressed anti-dsDNA production.

To further explore the role of cCD40L+CD4+ T cells in lupus autoimmunity, specially adapted in vitro protocols were used to study the effect of preformed cCD40L on B cell biology. Intriguingly, the results revealed that cCD40L+ T cells with memory phenotype (CD44highCD45RBlowCD62low) expressed CD40L at higher membrane density compared with naive cCD40L+CD4+ T cells following PMA/ionomycin stimulation. Furthermore, it was evident that most B cell responses were sustained only with optimal CD40 receptor occupancy by CD40LhighCD4+ T cells but not by CD40LlowCD4+ T cells (with preformed cCD40L stimulated in the presence of CHX). This is important in future attempts to identify the mechanism by which cCD40L+ T cells could contribute to lupus autoimmunity. Thus although these cells may not be capable of inducing responses in B cell when activated, they might induce myeloid-derived dendritic cells, which can in turn expand T cell responses to nucleosomal Ags (40).

The promotion of B cell survival from naive, or immunized, BWF1 mice after coculture with mitogen-stimulated T cells, but not with unstimulated BWF1 T cells, was blocked by anti-CD40L, confirming the significance of CD40-CD40L interaction for B cell survival. Interestingly, blockade of CD40-CD40L interaction in CHX-treated cells only partially prevented the survival of B cells from immunized mice. This may suggest a role for additional pathways in B cell rescue when the receptor occupancy by the cCD40L+ T cells is suboptimal. Previous studies have shown that cross-linking of integrin molecules LFA-1 and VLA-4 with their coreceptors ICAM-1 and VCAM-1, or cross-linking of CD21 with CD23 on FDC could rescue B cells from apoptosis (41, 42, 43). However, in experiments aimed at studying the effect of cCD40L ligation on T/B cell aggregation, we did not observe significant enhancement of aggregation after coculture of B cell with CHX-treated activated T cells.

In conclusion, naive cCD40L+ T cells observed in the spleen of BWF1 mice were able to rapidly up-regulate membrane CD40L after stimulation and promote the survival of B cells. Self-reactive B cells that arise by somatic mutations after Ag stimulation may receive signals to survive and produce Abs from T cells expressing low levels of CD40L. Further studies on molecular mechanisms of CD40L regulation in lupus to pinpoint the possible defect in CD40L regulation and the primary target cells influenced by these T cells may give a better understanding of mechanisms of lupus disease.


   Footnotes
 
1 This work was supported by the Arthritis Research Campaign. Back

2 Address correspondence and reprint requests to Dr. Rizgar A Mageed, The Kennedy Institute of Rheumatology, 1 Aspenlea Road, Hammersmith, London W6 8LH, U.K. Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; CD40L, CD40 ligand; CHX, cycloheximide; PDBu, phorbol-dibutyrate; PC, phosphorylcholine; 7-AAD, 7-amino actinomycin D; KLH, keyhole limpet hemocyanin; MFI, mean fluorescence intensity; BCR, B cell receptor; mBSA, methylated BSA; TA, terminal arterioles; PALS, periarteriolar lymphoid sheath; mCD40L+, membrane CD40L+; cCD40L+, cytoplasmic CD40L+. Back

Received for publication March 16, 2000. Accepted for publication July 17, 2000.


   References
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 Results
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