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The Journal of Immunology, 1998, 161: 729-739.
Copyright © 1998 by The American Association of Immunologists

Anti-CD40L Accelerates Renal Disease and Adenopathy in MRL-lpr Mice in Parallel with Decreased Thymocyte Apoptosis1

Jennifer Q. Russell*, Thomas Mooney*, Philip L. Cohen{ddagger}, Bruce MacPherson{dagger}, Randolph J. Noelle§ and Ralph C. Budd2,*

* Division of Immunobiology and {dagger} Department of Pathology, University of Vermont College of Medicine, Burlington, VT 05405; {ddagger} Division of Rheumatology, University of North Carolina School of Medicine, Chapel Hill, NC 27599; and § Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03756


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The CD40/CD40L (CD40 ligand) axis regulates several interactions between T cells and B cells. Blocking of CD40 engagement by CD40L inhibits Ig class switch by B cells as well as diminishes T cell response to an immunizing Ag. For these reasons, disruption of CD40/CD40L interactions by anti-CD40L administration or by genetic disruption of CD40L has ameliorated a variety of autoimmune conditions. More recent findings suggest that a direct signal can be transmitted to T cells via their expressed CD40L, which can costimulate proliferation with CD3 or promote germinal center formation. It is therefore possible that treatment with anti-CD40L Ab might produce a different outcome than observed in genetically CD40L-deficient mice. In this regard, we observe that in contrast to the genetic deletion of CD40L in MRL-lpr mice, which diminishes autoimmune disease but has little effect on adenopathy, administration of anti-CD40L to MRL-lpr mice accelerates both of these parameters. This difference appears to result from anti-CD40L actively delivering a signal that inhibits T cell apoptosis in lpr mice. This was confirmed by in vitro studies demonstrating that CD40L cross-linking on lpr thymocytes inhibited apoptosis and surface TCR down-modulation induced by CD3 ligation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mice homozygous for a retroviral disruption of the fas gene, designated lpr/lpr (lpr),3 develop an age- and thymus-dependent accumulation of T cells bearing an unusual phenotype, TCR-{alpha}ß+ CD4-CD8- B220+ (1, 2). In the near absence of Fas protein in lpr mice, peripheral deletion of T cells in response to superantigens is delayed (3, 4). The lpr mutation potentiates an autoimmune diathesis in the MRL/MpJ (MRL) strain, such that MRL-lpr mice develop a lupus-like syndrome that includes glomerulonephritis and IgG autoantibody production (5). Both T and B cells are required for the development of lupus in MRL-lpr mice. Treatment of these animals with Abs to Thy-1 or CD4 abrogates anti-DNA production and glomerulonephritis (6, 7). Similarly, the contribution of B cells is illustrated in either genetically B cell-deficient/lpr mice which manifest diminished renal disease (8), or in chimeric mice in which autoantibody production was from lpr but not +/+ B cells (9).

The generation of IgG Ab responses to conventional thymus-dependent Ags is dependent upon contact between T and B cells. This interaction requires binding of CD40 ligand (CD40L), a type II membrane protein transiently expressed on activated helper T cells, to CD40 on B cells, which provides a signal for Ig synthesis and class switching (10, 11). Mice or humans deficient in CD40 or CD40L are unable to promote an Ig class switch from IgM to IgG (12, 13, 14, 15, 16, 17, 18, 19, 20). This results in the presence of normal or increased levels of serum IgM and absent or decreased levels of IgG, IgA, and IgE. Stimulation of B cells by CD40 in conjunction with Ig engagement can also rescue them from Fas-induced apoptosis (21).

Preventing CD40/CD40L ligation abrogates a variety of autoimmune disorders including collagen-induced arthritis (22), experimental allergic encephalomyelitis (23), acute and chronic graft-vs-host disease (24), and many of the lupus-like features in (SWR x NZB)F1 mice (25) and in (NZB x NZW)F1 mice (26). While in some cases this was believed to be due to blocking of B cell function, it became apparent that T cell function was also profoundly diminished by blocking CD40. Ag immunization of CD40L-deficient mice (27), or of normal mice with concomitant administration of anti-CD40L Ab (28), greatly reduced T cell response to the Ag. Some of these effects appeared to be attributable to the lack of CD40-induced expression of B7-1 and B7-2 by B cells (29). Findings in other reports were more consistent with a direct signal delivered to T cells via CD40L. For example, administration of soluble CD40-Fc{gamma}1 to CD40-deficient mice restored germinal center formation, suggesting that direct engagement of CD40L on T cells was responsible for this effect (30). Another study demonstrated that anti-CD40L in vitro could costimulate proliferation of CD3-activated T cells (31).

Given the observations that CD40L might directly signal T cells, it is conceivable that administration of anti-CD40L Ab in vivo might produce considerably different outcomes of T cell function than those observed in CD40L-deficient mice. This appeared to be the case with our observations in MRL-lpr mice that received anti-CD40L. In contrast to recent findings in CD40L-deficient/lpr mice that manifested decreased autoimmune disease and little change in adenopathy (Ref. 32; and Dr. J. Craft, personal communication), we observed greatly accelerated disease and enhanced adenopathy. The latter is due, not to increased proliferation of lymphoid cells, but rather to their decreased rate of apoptosis. The findings suggest that CD40L can mitigate apoptotic signals in T cells.


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

MRL-lpr/lpr (lpr) mice were bred in the animal facilities at The University of Vermont College of Medicine from original breeding pairs obtained from The Jackson Laboratory (Bar Harbor, ME).

Anti-CD40L treatment

Eight-wk-old female MRL-lpr mice received either hamster monoclonal IgG anti-murine CD40L, MR1 (33), or control hamster IgG (ICN Pharmaceuticals, Costa Mesa, CA) at 250 µg i.p. twice weekly for 3 wk. In experiment 1, eight mice were treated per group, and one mouse from each group was euthanized after weeks 4 and 5 for analysis of lymphoid tissues. In experiment 2, 12 mice were treated in each group, and one mouse from each group was euthanized at weeks 2.5, 4, and 5 for analysis of lymphoid tissues and renal histology. The remaining six mice in experiment 1, and nine mice in experiment 2, were monitored for survival and quantitation of proteinuria using Chemstrip (Boehringer Mannheim Diagnostics, Indianapolis, IN) over an 18-wk (Expt. 1) or 20-wk (Expt. 2) period. Mice that achieved maximal 3+ proteinuria and weight loss were considered terminal and euthanized. Serum samples were taken approximately every 2 wk. Two additional experiments (Expts. 3 and 4) containing four female MRL-lpr mice per group were performed using the same protocol to confirm the lymphoid findings observed in the first two experiments. Finally, a fifth experiment was performed to determine whether prolonged anti-CD40L treatment could alter serum levels of total IgG as well as IgG autoantibodies. In this protocol, four MRL-lpr mice per group received the same initial biweekly Ab treatment for 3 wk followed by once weekly administration for an additional 12 wk.

Kidney histology

Fresh tissues were fixed in buffered 10% (v/v) paraformaldehyde for 24 h, washed in 70% (v/v) ethanol, and embedded in paraffin blocks. Serial kidney tissue sections were cut, fixed on slides, and stained with either hematoxylin and eosin (H & E) or periodic acid-Schiff (PAS) to assess, respectively, renal pathology. Sections were scored in a blinded manner by a renal pathologist (B.M.P.). A 0 to 2+ scale was used to quantitate severity for glomerular cellularity, interstitial inflammation, nuclear debris, tuft necrosis, basement membrane thickening, and sclerosis. Scoring was performed on 40 glomeruli from each mouse (20 glomeruli in each of two sections). In addition, the number of glomerular nuclei were counted in 10 separate glomeruli from each section stained with either H & E or PAS.

Quantification of serum Igs and autoantibody levels by ELISA

Sera from representative mice in both experiments were taken at the times indicated and analyzed for levels of total IgG, IgG1, and IgM. In addition, serum levels of total IgG and IgG1 autoantibodies to ssDNA, Sm Ag, and chromatin, as well as IgM rheumatoid factor, were determined by ELISA as previously described (34).

Abs and flow cytometry

Monoclonal anti-murine CD8{alpha} conjugated to phycoerythrin was purchased from Caltag Labs (Burlingame, CA). Monoclonal anti-murine CD4 conjugated to Red613 was purchased from Life Technologies (Gaithersburg, MD). mAb to mouse TCR-{alpha}ß, clone H57–597 (35), was purified from mouse ascites on HiTRAP protein G columns (Pharmacia Biotech, Piscataway, NJ) and then conjugated to fluorescein (Sigma Chemical, St. Louis, MO) using established methods (36). Fluorescein-conjugated Ab was purified from reaction components by chromatography on PD-10 columns (Pharmacia Biotech). Biotinylated anti-{kappa}-chain mAb, clone 187.1, was the kind gift of Dr. Karen Newell (The University of Vermont, Burlington, VT).

Single cell suspensions were made by homogenizing tissues in RPMI 1640 medium (Life Technologies) supplemented with 5% (v/v) bovine calf serum (HyClone Laboratories, Ogden, UT). Cells excluding trypan blue were counted. For flow cytometry, 106 cells were incubated in 0.1 ml PBS containing 0.5% BSA Fraction V, 0.001% (w/v) sodium azide (Sigma), and the indicated Abs, each at 3 µg/ml, at 4°C for 30 min. After washing with PBS-azide, cells were fixed in 1% (v/v) methanol-free formaldehyde (Ted Pella, Reading, CA) in PBS-azide. Samples were stored at 4°C until analysis with a Coulter Elite flow cytometer calibrated using DNA check beads (Coulter, Hialeah, FL). Data were gated using Elite software by forward and side light scatter. Negative controls were set by using isotype-matched Ig directly conjugated to fluorochromes (Caltag). To measure the surface expression of CD40L using MR1 mAb, thymocytes and LN cells were analyzed either when freshly isolated or after 4-h stimulation at 37°C in medium containing PMA (10 ng/ml) plus ionomycin (250 µg/ml).

TUNEL assay for apoptosis

Cells were initially stained for expression of TCR-{alpha}ß, CD4, and CD8 and then fixed for 15 min in 1% formaldehyde. Cell membranes were then permeabilized for 15 min using 70% ethanol at 4°C. Samples were incubated at 37°C for 1 h in 50 µl containing 10 U terminal deoxynucleotidyltransferase and 0.5 nM d-UTP-biotin (Boehringer Mannheim) (37, 38). Specimens were washed twice with PBS/1%BSA and incubated with a 1:50 dilution of streptavidin-tricolor (Caltag) at 4°C for 30 min. Cells were washed twice and analyzed by flow cytometry. Negative controls consisted of staining of cells with the same protocol but in the absence of d-UTP-biotin. Positive control staining for apoptosis was determined using thymocytes from mice that received 2 mg i.p. of dexamethasone 18 h previously.

In vitro anti-CD40L ligation of thymocytes

Single cell suspensions of thymocytes from 10-wk-old MRL-lpr mice were placed in RPMI 1640/5%FCS. Activation conditions included either control hamster IgG or anti-CD40L (10 µg/ml) for 20 min followed by cross-linking with goat anti-hamster IgG (50 µg/ml, Caltag) for 20 min and finally addition of anti-CD3 (500A2, 10 µg/ml). Timing was begun at the addition of anti-CD3, and cells were analyzed either freshly isolated or 3.5 h and 5.0 h after activation. Thymocytes were stained for expression of CD4, CD8, TCR-{alpha}ß, fixed, and then stained by the TUNEL assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-CD40L Ab treatment of MRL-lpr mice accelerates glomerulonephritis and decreases survival

Given the existence of a wide variety of IgG autoantibodies in MRL-lpr mice, we attempted to diminish their serum levels using administration of anti-CD40L in vivo, while monitoring proteinuria and survival. Details of the five separate experiments are given in the Materials and Methods. In brief, the first four experiments consisted of 3 wk of twice weekly anti-CD40L administration followed by a total 18-wk (Expt. 1) or 20-wk (Expt. 2) period of monitoring. A fifth experiment examined whether prolonged (15 wk) anti-CD40L treatment might more efficiently suppress IgG autoantibody formation.

In striking contrast to the findings in the (SWR x NZB)F1 mice (25), as well as a recent report of diminished disease in CD40L-deficient/lpr mice (32), anti-CD40L administration to MRL-lpr mice resulted in accelerated disease and adenopathy, as well as decreased survival. The onset of proteinuria was earlier in MRL-lpr mice receiving anti-CD40L. The first experiment is shown in Figure 1GoA and illustrates the percentage of mice that had achieved 3+ proteinuria during the 18-wk period. By 10 wk, four of the six mice (67%) receiving anti-CD40L had achieved 3+ proteinuria, whereas only two of six control mice that received hamster IgG (33%) manifested this degree of proteinuria. The increased proteinuria in the anti-CD40L group persisted throughout the 18-wk period of observation. The second experiment with 12 mice per group showed very similar accelerated proteinuria over a 20-wk period in the mice receiving anti-CD40L (data not shown). These differences were statistically significant in both experiments by both the Wilcoxon signed rank test (p = 0.005) and t test (p = 0.002).



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  		FIGURE 1. Anti-CD40L treatment of MRL-lpr mice accelerates the development of proteinuria (A) and diminishes survival (B). Six 8-wk-old female MRL-lpr mice per group received 250 µg i.p. of either hamster IgG (dashed line) or anti-CD40L Ab (solid line) twice weekly for 3 wk and were then monitored over a total period of 18 wk. Statistical analysis was by Wilcoxon signed rank test for proteinuria and log rank survival for the survival studies. Similar results were observed in a second 20-wk study using nine MRL-lpr mice per group.

 
Survival was also diminished in the group of MRL-lpr mice receiving anti-CD40L. As shown in Figure 1GoB, by the end of the 18-wk study period in experiment 1, only two of six mice were alive in the anti-CD40L treatment group, whereas all six mice that received hamster IgG survived. By log rank survival curve this was significant at p = 0.019. Similar findings, though somewhat less pronounced, were observed in the second experiment.

The more rapid onset of proteinuria in MRL-lpr mice receiving anti-CD40L was reflected in histologic evidence of renal injury, as defined by the number of glomerular nuclei (Fig. 2GoA), as well as increased glomerular cellularity (Fig. 2GoB) and glomerular inflammation, with or without nuclear debris (Fig. 2GoC). The glomerular histology was assessed in a blinded manner by a renal pathologist. A statistically significant increase in the number of glomerular nuclei was observed at all three time points (2.5 wk, p = 0.019; 4 wk, p = 0.043; and 5 wk, p = 0.005 by t test) in the mice receiving anti-CD40L, while a significant increase in the severity of glomerular cellularity and inflammation was seen in the same mice at 2.5 wk (p = 0.001 and 0.001, respectively) and 5 wk (p = 0.001 and 0.002, respectively). These findings correlate well with the accelerated proteinuria that was observed with anti-CD40L administration.



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  		FIGURE 2. Increased glomerular injury in MRL-lpr mice receiving anti-CD40L. In Expt. 2, beginning at 8 wk of age, 12 female MRL-lpr mice received 3 wk of biweekly hamster IgG (HIgG) or anti-CD40L, and one mouse per group was euthanized at weeks 2.5, 4, and 5. Both kidneys of each mouse were removed and fixed, and sections were stained with H & E or PAS. A, Absolute counts and means (horizontal bar) of number of nuclei for each of 10 glomeruli for each mouse. B, C, Mean (± SEM) score of 40 glomeruli of each mouse analyzed for (B) increase in glomerular cellularity, or (C) severity of glomerular inflammation using a 0 to 2+ scale of severity. Statistical significance by p value (t test) is shown above each time point.

 
Anti-CD40L treatment does not significantly alter autoantibody production in MRL-lpr mice

Since CD40 is required for Ig class switching from IgM to IgG, mice or humans deficient in CD40L manifest greatly diminished serum levels of IgG and frequently elevated levels of IgM (13, 18). In experiments 1 and 2, as MRL-lpr mice received Ab during only the first 3 wk of the study, it is perhaps not surprising that serum levels of Ig isotypes were largely comparable between the two treatment groups, as were levels of IgG and IgM autoantibodies to ssDNA, Sm Ag, chromatin, and rheumatoid factor (data not shown). However, even with anti-CD40L treatment for up to 12 wk, as in experiment 5, there was still no consistent statistically significant difference in total serum IgG1 or IgM (Fig. 3Go, A and B). In a similar manner, although serum levels of IgG1 (Fig. 3Go, D–F), IgG2a, and IgG3 (data not shown) autoantibodies to ssDNA and Sm Ag, but not chromatin, were slightly decreased in mice receiving anti-CD40L and the corresponding IgM autoantibodies somewhat increased (Fig. 3GoG), these did not achieve statistical significance. This suggests that the CD40/CD40L axis may have little impact on the development of several types of autoantibodies in MRL-lpr mice. This is in agreement with CD40L-deficient/lpr mice, which also were reported to manifest unaltered levels of certain autoantibodies (32).



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  		FIGURE 3. Serum levels of total IgG1 and certain IgG1 autoantibodies are diminished, and corresponding IgM levels are increased in MRL-lpr mice receiving prolonged administration of anti-CD40L. Shown are the serum levels of the indicated Ab measured by ELISA from individual female MRL-lpr mice of experimenet 5 that received either control hamster IgG (closed circles) or anti-CD40L (open circles) twice weekly for the first 3 wk, followed by weekly Ab treatment for an additional 12 wk. Note during the later weeks that anti-CD40L treatment caused diminished levels of total IgG1 and IgG1 autoantibodies to ssDNA and to Sm Ag, but not chromatin. The corresponding levels of IgM were elevated in the same mice.

 
Anti-CD40L treatment promotes adenopathy and the expansion of TCR-{alpha}ß+ cells in the thymus and LN of MRL-lpr mice

A remarkable expansion of T cell numbers occurred rapidly in both the thymus and LN during the first few weeks of anti-CD40L treatment, as is summarized for experiments 1 and 2 in Table IGo. This was highly statistically significant for both the thymus (p = 0.005) and LN (p = 0.007). As shown in Figure 4Go (bottom row), as early as 2.5 wk after initiating anti-CD40L administration, thymus cellularity was considerably increased by 1.5- to 2-fold, and this persisted at 5 wk, at a point when anti-CD40L treatment had ended 2 wk previously.


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  		Table I. Increased lymphoid cell number and peripheral TCR-{alpha}ß+ CD4-CD8- cells in MRL-lpr mice receiving anti-CD40La

 


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  		FIGURE 4. Anti-CD40L increases thymus cellularity and promotes the appearance of TCR-{alpha}ß+ thymocytes in all subsets. Shown are FACS profiles of the thymuses from 8-wk-old female littermate MRL-lpr mice that had received either hamster IgG (upper row) or anti-CD40L (middle row) twice weekly for 2.5 wk. Percentages of thymocytes in each gate are indicated by the number inserts. Note the slightly increased proportion of mature single positive thymocytes as well as the increased percentage expressing TCR-{alpha}ß with anti-CD40L treatment. The bottom row illustrates thymocyte absolute numbers in the subsets indicated. Hatched bars represent cell numbers in mice that received control hamster IgG, whereas solid bars correspond to cell numbers in anti-CD40L-treated mice. These findings were highly consistent over the course of five separate experiments using twice weekly treatment for 3 wk and examining tissues at intervals ranging from 1 to 5 wk.

 
An analysis of the phenotypes of the thymocytes revealed a slight but consistently enhanced proportion of mature single positive TCR-{alpha}ß+ thymocytes with anti-CD40L treatment (Fig. 4Go, compare top two rows). Although the proportions of CD4+CD8+ and CD4-CD8- thymocytes were correspondingly somewhat diminished with anti-CD40L, due to the pronounced increased thymic cellularity, the absolute numbers in these subsets were nonetheless considerably increased. The suggestion that a larger proportion of mature thymocytes emerged with anti-CD40L treatment is supported by an increase in the expression of TCR-{alpha}ß among all thymocyte subsets (Fig. 4Go, middle row). A complete listing of thymocyte and LN cell numbers and subset percentages is shown for experiments 1 and 2 in Table IGo. The total number of thymocytes was statistically increased with anti-CD40L treatment (p = 0.005) as well as the percentage of CD4+CD8- thymocytes (p = 0.027), while the proportional increase in CD4-CD8+ thymocytes was just below significance (p = 0.064).

Cellularity differences were even more striking in the LN. Anti-CD40L treatment produced up to a fivefold increase in LN cell number by 5 wk (Fig. 5Go, bottom row). In contrast to the thymus where the proportions of mature CD4+ and CD8+ T cells were increased, the expanding LN cell population with anti-CD40L manifest decreased proportions (though increased absolute numbers) of mature T cells, due to the dilution by TCR-{alpha}ß+ CD4-CD8- cells (Fig. 5Go, middle row). Over the course of experiments 1 and 2, both the total number of LN cells (p = 0.007) and the proportion of TCR-{alpha}ß+ CD4-CD8- cells (p = 0.024) were statistically increased in the group that received anti-CD40L (Table IGo).



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  		FIGURE 5. Dramatically increased T cell accumulation in LN of MRL-lpr mice receiving anti-CD40L. LN are from the same mice whose thymuses are shown in Figure 4Go. Although both the CD4+ and CD8+ subsets were increased in absolute numbers with anti-CD40L treatment, their percentages were diminished due to the preferential expansion of the CD4-8- subset, which contained an increased proportion of TCR-{alpha}ß+ cells. The findings were very consistent over the course of five separate experiments.

 
A similar though not quite as profound increase of splenic B cells was also observed with anti-CD40L treatment of lpr mice. As shown in Figure 6Go, both the proportion as well as absolute number of splenic B cells was increased. The increase in B cell number was not manifest as quickly as were the increases of thymocyte and LN T cell numbers. Whereas the changes in thymocyte and LN cellularity was apparent as early as 1 wk in some experiments, the increase in splenic B cells was not observed until at least wk 3 or 4 of anti-CD40L treatment. The effects of anti-CD40L on lpr adenopathy were not limited to the MRL background since similar accelerated adenopathy was observed in C57BL/6-lpr mice that received anti-CD40L biweekly for 5 wk (data not shown). Moreover, these differences were not likely related to alterations in Ag presentation since surface levels of MHC class I, class II, B7-1, and B7-2 were identical in the two groups of mice (data not shown).



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  		FIGURE 6. Accumulation of B cells in MRL-lpr mice receiving anti-CD40L. Spleens are from the same mice as shown in Figures 4Go and 5Go that received anti-CD40L twice weekly for 3 wk and studied at the times indicated above each column. Shown in the top two rows are the proportions of splenocytes staining with anti-{kappa}-chain Ab that were in the CD4-8- gate. The bottom row shows the absolute B cell number. The findings were consistent throughout the five experiments.

 
Anti-CD40L decreases apoptosis in lpr thymocytes

It was not clear from the above findings whether the increased lymphoid cellularity with anti-CD40L treatment resulted from increased proliferation and/or decreased apoptosis. This was examined, using propidium iodide to measure cell cycling and the TUNEL assay to quantitate apoptosis. Cell cycle analysis by propidium iodide revealed only low levels of cell cycling in the thymus, and LN and was no different with anti-CD40L or hamster IgG. Similarly, in vitro culture of normal or lpr LN cells with anti-CD3 demonstrated no augmented proliferation with the addition of anti-CD40L, either soluble or immobilized (data not shown). In contrast, the level of apoptosis in the thymus, as revealed by the TUNEL assay, was considerably lower in the anti-CD40L treatment group. Figure 7GoA shows the percentage of TCR-{alpha}ß+ thymocytes in each subset that bears degraded DNA (numbers in parentheses). Anti-CD40L treatment resulted in diminished levels of degraded DNA among TCR-{alpha}ß+ thymocytes in all four subsets defined by CD4 and CD8. This was frequently at least twofold less than observed in control mice. These differences in thymocyte apoptosis were not detectable in the LN, perhaps due to the intrinsically low levels of apoptosis at this site (Fig. 7GoB). This would suggest that the pronounced increased cellularity of the LN with anti-CD40L might have resulted from increased thymic output of precursors of TCR-{alpha}ß+ CD4-CD8- cells rather than in situ expansion of these cells in LN.



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  		FIGURE 7. Decreased level of thymocyte apoptosis with anti-CD40L treatment. Following 2 wk of twice weekly treatment with hamster IgG (upper row) or anti-CD40L (lower row), thymocytes (A) or LN cells (B) were incubated in serum-free medium at 37°C for 4 h. Cells were then surface stained for expression of CD4, CD8, and TCR-{alpha}ß followed by staining for degraded DNA using the TUNEL assay. Negative control staining was on the thymocytes from hamster IgG-treated mice and were surface stained with hamster IgG-FITC, and TUNEL staining was performed with all steps except in the absence of biotin-dUTP. Positive TUNEL control staining was on thymocytes from mice that received 2 mg of dexamethasone 18 h previously. Number inserts without parentheses indicate the proportion of total cells expressing TCR-{alpha}ß+ with or without degraded DNA. Number inserts within parentheses indicate the percentage of TCR-{alpha}ß+ cells containing nicked DNA within each subset defined by CD4 and CD8.

 
To further examine whether the delayed thymocyte apoptosis resulting from anti-CD40L was an in vivo artifact, an analogous study was performed on lpr thymocytes in vitro. Thymocyte suspensions were placed in serum-free medium, and anti-CD40L or hamster IgG was added, followed by cross-linking. Anti-CD3 was then added, and apoptosis was quantitated by TUNEL assay at 3.5 or 5.0 h. Figure 8Go shows that, compared with the hamster IgG control, thymocytes pretreated with anti-CD40L before CD3 ligation manifested strikingly less down-regulation of TCR as well as decreased apoptosis, which was present primarily in the TCRlow subset. Table IIGo shows that these findings were highly consistent in three experiments and statistically significant (p = 0.016).



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  		FIGURE 8. Anti-CD40L cross-linking inhibits in vitro CD3-induced apoptosis of thymocytes. MRL-lpr thymocytes were placed in serum-free medium with either control hamster IgG (HIgG) or anti-CD40L followed by goat-anti-hamster IgG and then anti-CD3. At the times indicated, cells were stained for expression of CD4, CD8, TCR-{alpha}ß, fixed, and then stained by the TUNEL assay. Numbers in the CD4 vs CD8 staining indicate the percentage of total cells in each subset. Numbers in the TUNEL histograms indicate the total percentage of cells bearing degraded DNA. Note the lack of TCR down-modulation in the presence of anti-CD40L as well as decreased apoptosis, which was statistically significant over the course of three similar experiments (p = 0.016, see Table IIGo).

 

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  		Table II. Decreased apoptosis in vitro of lpr thymocytes following CD40L ligationa

 
MRL-lpr mice have decreased expression of CD40L

Although thymocyte enlargement with anti-CD40L treatment in vivo has been observed in other strains of normal mice (39), LN expansion has not been reported. The striking lymphoid hyperplasia seen with anti-CD40L treatment of MRL-lpr mice might reflect an increased expression of CD40L by lpr T cells. This was not the case. In both MRL-lpr and MRL +/+ mice, unstimulated thymocytes revealed similarly low levels of CD40L expression (Fig. 9GoA). Following activation with PMA plus ionomycin for 4 h, there was a substantially increased expression of CD40L on MRL +/+ thymocytes that was more pronounced on the CD4+CD8- subset compared with the CD4-CD8+ or other subsets, in agreement with previous findings (39). In contrast, the activated CD4+CD8- thymocytes of MRL-lpr mice expressed considerably lower levels of CD40L, as did the CD4-CD8+ subset (Fig. 9GoA). A similar difference of CD40L expression was observed in the same subsets of LN cells between MRL +/+ and MRL-lpr mice (Fig. 9GoB). Of particular note is that the TCR-{alpha}ß+ CD4-CD8- subset of lpr LN cells expressed low to negligible levels of CD40L, either unstimulated or following activation with PMA plus ionomycin. This creates a seeming paradox in that CD40L is not expressed by the lpr LN cell type that accumulates to the greatest degree with anti-CD40L treatment. The explanation for this disparity may lie in the realization that the lineage of TCR-{alpha}ß+ CD4-CD8- LN cells in lpr mice derives from mature T cells, primarily of the CD8+ subset (40, 41).



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  		FIGURE 9. Decreased expression of CD40L by lpr T cells. Thymocytes (A) or LN cells (B) from MRL +/+ (upper row) or MRL-lpr (lower row) mice were analyzed either unstimulated or after stimulation for 4 h with PMA plus ionomycin. Cells were then stained for expression of CD4, CD8, TCR-{alpha}ß, and CD40L. Shown is the expression of TCR-{alpha}ß vs CD40L on the subsets indicated. Number inserts indicate the percentage of positive cells in each quadrant. Only a small proportion of CD4-8+ cells of either strain expressed CD40L. However, the CD4+8- subset of MRL +/+ T cells manifested significant up-regulation of CD40L within 4 h of stimulation, and this was considerably diminished in the same subset of MRL-lpr mice. As can be inferred from the staining of lpr total LN (B), the major subset of TCR-{alpha}ßintermediate CD4-8- cells was largely devoid of CD40L expression even after mitogen activation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current findings show that, in marked contrast to CD40L-deficient/lpr mice, treatment of MRL-lpr mice with anti-CD40L Ab manifested accelerated adenopathy, glomerulonephritis, and death. Although IgG autoantibody production persisted to various degrees with both methods of CD40 signal blocking, the CD40L-deficient/lpr mice developed less disease and little difference in adenopathy compared with standard MRL-lpr mice (32). The collective observations from both systems suggest that in CD40L-deficient/lpr mice the resulting phenotype is due to the actual disruption of the CD40/CD40L axis, whereas administration of anti-CD40L confers an active signal. Our data suggest that this CD40L signal manifests as a partial inhibition of T cell apoptosis beyond that which results from the absence of Fas expression in lpr mice.

The notion that CD40L itself may act as a direct signaling molecule, beyond its capacity to engage CD40, has received only modest attention. Cayabyab et al. (31) reported that CD40+ P815 cells could costimulate in vitro with CD3 to promote proliferation of CD4+ cells. Using an in vivo model, administration of soluble CD40-Fc{gamma} to CD40-deficient mice restored germinal center formation, although it did not induce Ig class switch (30). This was interpreted as the ability of soluble CD40-Fc{gamma} to activate T cells directly through CD40L to promote germinal center formation. A further study showed that CD40L-deficient mice have a defect in T cell priming that could not be attributed to defective APC function, since CD40L-/- APC could strongly stimulate CD40+/+ T cells (27). Although these three reports did not examine apoptosis of T cells, they are consistent with the concept of direct signal transduction via CD40L.

A further study reported that anti-CD40L treatment of mice did prevent the deletion of thymocytes bearing self-reactive TCR-Vß (39). However, this was also observed in CD40L-deficient mice, and both types of mice manifested diminished thymic expression of B7-2. It was consequently viewed that diminished signaling via B7-2 was in part responsible for the decreased apoptosis. While this may be accurate, the findings are also consistent with a model in which direct signaling through CD40L may also inhibit thymocyte apoptosis. Our observation that in vitro treatment of thymocytes with anti-CD40L prevented CD3-induced TCR down-modulation as well as apoptosis suggests that CD40L signaling might have the effect of decreasing the intensity of TCR signaling on thymocytes. Ligation of CD40L also has been shown to up-regulate expression of cell adhesion molecules, such as ICAM and CD44H (42, 43). Lymphocytes can be protected from apoptosis when they are in contact with various stromal cells, such as fibroblasts (44, 45). Conceivably, part of the rescue from apoptosis by CD40L stimulation may result from secondary promotion of T cell adhesion to stromal components.

Anti-CD40L treatment blocks progression of a variety of T cell-mediated autoimmune diseases, including collagen-induced arthritis (22), experimental allergic encephalomyelitis (23), chronic graft-vs-host disease (24), as well as other murine models of lupus such as (SWR x NZB)F1 (SNF1) or (NZB x NZW)F1 mice (25, 26). Anti-CD40L administration during chronic graft-vs-host disease blocked production of IgG anti-chromatin and rheumatoid factor (24). In SNF1 mice, as few as three injections of anti-CD40L given to pre-nephritic mice at 3 mo of age markedly reduced the incidence of nephritis for as long as 12 mo. In addition, autoantibody production was inhibited by this anti-CD40L regimen. This stands in marked contrast to the persistence of some IgG autoantibodies in both CD40L-deficient/lpr mice, and the lpr mice given anti-CD40L in this study. In the case of CD40L-deficient/lpr mice, IgG autoantibodies to small nuclear ribonucleoproteins (snRNPs) were persistent whereas development of anti-DNA Abs and rheumatoid factor was absent (32). With prolonged anti-CD40L treatment of MRL-lpr mice, we also observed moderate decreases in serum IgG1 autoantibodies to ssDNA, Sm Ag, but not chromatin, particularly at later times. Compared with the similar findings from CD40L-deficient/lpr mice, these results may partly reflect known intrinsic abnormalities in lpr B cells (34). Another contributing factor may be the increase in B cell number that was observed with anti-CD40L treatment. This was unexpected and may be secondary to the enormous increase in T cells with anti-CD40L treatment, which could provide augmented help to B cells, to enable them to overcome the partial CD40 block. The resulting increase in splenic B cells may have partly compensated for inhibition of IgG class switching and Ig production, by blocking CD40 interactions, and resulted in maintenance of certain IgG autoantibodies.

Although diminished negative selection has been observed in the thymocytes from either CD40L-deficient mice or mice that received anti-CD40L (39), peripheral lymphadenopathy was not observed, nor was it noted in the above mentioned autoimmune disorders that were treated with anti-CD40L. A model consistent with these earlier findings as well as the augmented adenopathy we observed might be that ligation of CD40L on thymocytes inhibits apoptosis and leads to increased emigration of mature T cells to the periphery. Such an increased export of thymocytes might not be apparent in normal mice since apoptosis in the periphery occurs normally. Only in the absence of Fas would this increased T cell output become profoundly manifest. Along the same line of reasoning, lpr T cells manifest several signaling abnormalities, including increased p56fyn (46), increased phosphorylation of CD3{zeta} (47), as well as decreased cytokine production and proliferative capacity (48). Conceivably, these signaling aberrations may allow a direct CD40L signal to become more apparent than in normal T cells.

The model might also serve to explain two paradoxes. The first is that, although anti-CD40L diminished apoptosis in the thymus, with increased absolute numbers of mature CD4+ and CD8+ thymocytes, what actually accumulated in the periphery in most instances was preferentially the TCR-{alpha}ß+ CD4-CD8- T cell subset. The second paradox was that, unlike lpr mature T cells, lpr CD4-CD8- T cells do not express CD40L, even though they accumulate to a greater degree with anti-CD40L. Knowing that lpr CD4-CD8- T cells arise by active positive selection from mature T cells, particularly the CD8+ subset (40, 41), the increased outpouring of mature thymocytes to the periphery might provide a considerably increased precursor supply of T cells that would subsequently become CD4-CD8- upon receiving the necessary high intensity TCR signal (49).


   Acknowledgments
 
We thank Colette Charland for assistance with flow cytometry.


   Footnotes
 
1 This work was supported in part by grants from the National Institutes of Health (AR-33887 to P.L.C. and AI-36333 to R.C.B.). Back

2 Address correspondence to Dr. Ralph C. Budd, The University of Vermont College of Medicine, Given Medical Building C-303, Burlington, VT 05405. E-mail address: Back

3 Abbreviations used in this paper: lpr, lpr/lpr; MRL, MRL/MpJ; CD40L, CD40 ligand; LN, lymph node; PAS, periodic acid-Schiff; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling; SNF1, (SWR x NZB)F1; H & E, hematoxylin and eosin; Sm Ag, Smith antigen. Back

Received for publication August 6, 1997. Accepted for publication March 23, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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