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The Journal of Immunology, 2008, 181, 47-61
Copyright © 2008 by The American Association of Immunologists, Inc.
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*Lupus

CTL-Promoting Effects of CD40 Stimulation Outweigh B Cell-Stimulatory Effects Resulting in B Cell Elimination and Disease Improvement in a Murine Model of Lupus1

Roman Puliaev*,{dagger}, Irina Puliaeva*,{dagger}, Lisbeth A. Welniak{ddagger}, Abigail E. Ryan*,{dagger}, Mark Haas§, William J. Murphy{ddagger} and Charles S. Via2,*,{dagger}

* Pathology Department, Uniformed Services University of Health Sciences, Bethesda, MD 20814; {dagger} Research Service, Baltimore Veterans Affairs Medical Center, and Division of Rheumatology and Clinical Immunology, University of Maryland School of Medicine, Baltimore, MD 21201; {ddagger} Department of Microbiology and Immunology, University of Nevada School of Medicine, Reno, NV 89557; and § Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21287


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD40/CD40L signaling promotes both B cell and CTL responses in vivo, the latter being beneficial in tumor models. Because CTL may also limit autoreactive B cell expansion in lupus, we asked whether an agonist CD40 mAb would exacerbate lupus due to B cell stimulation or would improve lupus due to CTL promotion. These studies used an induced model of lupus, the parent-into-F1 model in which transfer of DBA/2 splenocytes into B6D2F1 mice induces chronic lupus-like graft-vs-host disease (GVHD). Although agonist CD40 mAb treatment of DBA->F1 mice initially exacerbated B cell expansion, it also strongly promoted donor CD8 T cell engraftment and cytolytic activity such that by 10 days host B cells were eliminated consistent with an accelerated acute GVHD. CD40 stimulation bypassed the requirement for CD4 T cell help for CD8 CTL possibly by licensing dendritic cells (DC) as shown by the following: 1) greater initial activation of donor CD8 T cells, but not CD4 T cells; 2) earlier activation of host DC; 3) host DC expansion that was CD8 dependent and CD4 independent; and 4) induction of acute GVHD using CD4-depleted purified DBA CD8+ T cells. A single dose of CD40 mAb improved lupus-like renal disease at 12 wk, but may not suffice for longer periods consistent with a need for continuing CD8 CTL surveillance. These results demonstrate that in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, CTL promotion is both feasible and beneficial and the CTL-promoting properties of CD40 stimulation outweigh the B cell-stimulatory properties.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CD40:CD154 signaling is an essential step in the initiation of both CD8+ CTL responses and T-dependent B cell responses (reviewed in Ref. 1). In lupus, excessive B cell activation and autoantibody production are CD4 T cell driven (2, 3, 4), and lupus T cells exhibit excessive CD154 expression (5, 6, 7). Therapeutic efforts in lupus aimed at blocking CD40:CD154 interactions using anti-CD154 mAb have been shown to retard disease in both murine and human lupus; however, serious thrombotic complications have limited this approach in humans (8).

Targeting of CD40 may nevertheless be of benefit in lupus. For example, CTL are well known to play an important role in tumor surveillance and in the elimination of intracellular pathogens (9, 10, 11, 12), and CD40 stimulation with an agonist mAb was shown to be beneficial in tumor models due to enhanced CTL activity (13, 14). CTL may also be important in limiting lupus-associated expansion of autoreactive B cells, as suggested by work demonstrating that both Fas and perforin (pfp)3 pathways are important in retarding disease expression in spontaneous (15, 16) and induced (17, 18) models of murine lupus. In human lupus, studies using PBL from monozygotic twins discordant for systemic lupus erythematosus are consistent with a pre-existing defect in cytolytic function (19). Taken together, these studies support the idea that CD8 CTL may be an unrecognized down-regulatory mechanism that could limit lupus expression. It is possible then that CD40 stimulation might be beneficial in lupus by inducing CTL that eliminate autoreactive B cells. Conversely, it is also possible the B cell-stimulatory properties of agonist CD40 mAb might exacerbate lupus.

A useful mechanistic model for the screening of agents with in vivo CTL-promoting activity in the setting of lupus-like disease is the parent-into-F1 (P->F1) model of graft-vs-host disease (GVHD). In this model, the transfer of normal parental CD4+ and CD8+ T cells into unirradiated normal F1 mice, e.g., C57BL/6->B6D2F1 results in the generation of donor CD8+ CTL specific for host MHC I (20, 21) that eliminate host splenic lymphocytes, particularly B cells, by 2 wk, resulting in a lymphopenic state termed acute GVHD. An interesting exception is the transfer of DBA/2 splenocytes into B6D2F1 mice, which does not result in acute GVHD, but instead results in a lupus-like chronic GVHD due to a combination of donor CD4 T cell allorecognition and help for host B cells and impaired donor CD8+ CTL effectors. The ensuing impairment in host B cell elimination permits host B cell expansion, autoantibody production, and eventually a lupus-like renal disease (22). Coadministration of agents that promote CTL, such as rIL-12, can correct the defective CTL response in DBA->F1 mice and convert disease phenotype from chronic lupus-like GVHD to CD8+ CTL-mediated acute GVHD (23, 24). Thus, in this model, agents that promote CTL in vivo can also prevent lupus. In this study, we used the DBA->F1 combination to test the usefulness of an agonist CD40 mAb in murine lupus by determining which of its two major properties, CTL promotion or B cell stimulation, would prevail. Our results indicate that although CD40 stimulation initially promotes both B cell expansion and CD8+ T cell activation, the CTL-promoting effects of anti-CD40 mAb outweigh the B cell-stimulatory effects and result in elimination of autoantibody-secreting B cells in the short-term and in improved renal disease long-term.


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

Male mice aged 6–8 wk were purchased from The Jackson Laboratory. Male C57BL/6J (B6) and DBA/2J (DBA) mice were used as donors, and B6D2F1 (BDF1) mice were used as recipients. All animal procedures were preapproved by the Institutional Animal Care and Use Committee at the Uniformed Services University of Health Sciences and at the University of Maryland School of Medicine.

Induction of GVHD

Single-cell suspensions of donor strain splenocytes were prepared in RPMI 1640, filtered through sterile nylon mesh, washed, and diluted to the desired concentration of viable (trypan blue excluding) cells/ml. Acute GVHD was induced using 50 x 106 unfractionated B6 splenocytes, and chronic GVHD was induced with either 12 x 106 B6 CD4+ (CD8+ T cell-depleted) splenocytes or 80 x 106 DBA splenocytes. Depletion of donor CD8+ or isolation of donor CD8+ T lymphocytes was performed using Dynabeads Mouse CD8 (Lyt 2) or Dynal Mouse CD8 Negative Isolation Kit, respectively, from Invitrogen, according to the manufacturer’s instructions. Flow cytometric analysis before cell transfer confirmed <1% contaminating CD8+ or CD4+ T cells. Cell suspensions were transferred via tail vein injection into normal, unirradiated F1 recipients. Control mice consisted of uninjected age- and sex-matched F1 mice.

In vivo reagents

Anti-mouse CD40 (FGK115, IgG1 agonistic mAb) was produced as ascites, as described previously (25). Endotoxin content was determined by the Limulus amebocyte lysate assay (QCL-1000; BioWhittaker) and was below the lower limit of detection (<3.13 EU/ml). The Ab was administered at a dose of 100 µg/mouse i.v. Dosing schedule is as described in the figure legends and text. Control mAb (ChromPure rat IgG; Jackson ImmunoResearch Laboratories) was administered at the same dose and schedule. Additional controls consisted of untreated acute and chronic GVHD mice, anti-CD40 mAb-treated normal F1 mice, and uninjected normal F1 mice. For experiments using purified donor CD8 T cells, anti-NK1.1 mAb (PK136 hybridoma; National Cell Culture Center) was given at 200 µg/mouse i.p. 2 days before cell transfer and on day 3 afterward.

Flow cytometric analysis

Spleen cells were first incubated with anti-murine Fc{gamma}RII/III mAb, 2.4G2, for 20 min, and then stained with saturating concentrations of FITC-conjugated, biotin-conjugated, PE-conjugated, Alexa Fluor 647-conjugated, or allophycocyanin-Alexa Fluor 750-conjugated mAb against CD4, CD8, B220, H-2Kd, I-Ad, H-2Kb, I-Ab, Fas (CD95), Fas ligand (FasL) (CD178), CD11c, CD80, CD21/CD35, and CD23 purchased from BD Pharmingen and Invitrogen. Biotinylated primary mAb were detected using either streptavidin-Alexa Fluor 350 or streptavidin-PE-Cy5 conjugates. Multicolor flow cytometric analyses were performed using a BD FACScan flow cytometer or BD LCRII flow cytometer (BD Biosciences). Monocyte populations were excluded on the basis of forward and side scatter. Lymphocytes were gated by forward and side scatter, and fluorescence data were collected for 10,000 cells. Studies of donor T cells were performed on 4,000–5,000 gated cells that were positive for CD4+ or CD8+ and negative for MHC class I of the uninjected parent. Gated donor T cells were analyzed for FasL expression and compared with uninjected strain-matched splenocytes. The percentage positivity for Fas, FasL, and CD80 for experimental groups was determined using a setting such that 95% of the relevant control cell population (uninjected parental or host strain) was negative.

For CFSE staining, suspensions of splenocytes were prepared, as described above, and resuspended at 50 x 106 cells/ml in PBS/0.1% BSA, and 5 µM of CFSE (Invitrogen) was added to the cell suspensions. Cells were mixed and incubated for 15 min at 37°C. Unbounded CFSE was quenched by the addition of 5 vol of ice-cold RPMI 1640/10% FCS and incubated on ice for 5 min. Cells were washed three times with RPMI 1640. FACS analysis of cells following CFSE labeling indicated a labeling efficiency 98–99%. Proliferation of transferred cells was visualized as incremental loss of CFSE fluorescence and was analyzed using the ModFit LT software (Verify Software House) at the times indicated. Calculation of divided cells was performed as described (26).

In vivo cytotoxic T cell activity

In vivo cytotoxic activity was determined using CFSE-labeled target cells, as described (27, 28, 29). Briefly, target cells consisted of both parental strain splenocytes loaded with CFSE, as described above, at two different concentrations, as follows: 0.5 µM for B6 targets (CFSElow) and 5 µM for DBA targets (CFSEhigh). Cell suspensions were irradiated at 2000 R, and F1 mice were injected i.v. with a 1:1 mixture of both target cell populations (1 x 107 cells each, 2 x 107 cells total). Target cell CFSE staining and parental cell ratios were verified by flow cytometric analysis before injection. At 5 h after injection of target cells, mice were sacrificed and splenocytes were analyzed by flow cytometry to determine the percentage of CFSElow and CFSEhigh target cells. Mice were tested individually, and the absolute number of each target cell population was calculated for each mouse based on the total spleen cell count multiplied by the percentage of positive cells determined by flow cytometry. The percent specific lysis of anti-host killing was determined relative to donor strain killing. For DBA->F1 mice, the formula was: % lysis = 100 – (((% B6 CFSElow experimental/% DBA CFSEhigh experimental)/(mean % B6 CFSElow normal F1 group/mean % DBA CFSEhigh normal F1 group)) x 100). For B6->F1 mice, the formula was: % lysis = 100 – (((% DBA CFSEhigh experimental/% B6 CFSElow experimental)/(mean % DBA CFSEhigh normal F1/mean % B6 CFSElow normal F1)) x 100). Substitution of the absolute target cell numbers for the percentage of target cell numbers in the preceding formulas did not significantly alter the results.

In vivo cytokine capture assay (IVCCA)

The IVCCA (BD Pharmingen) (30, 31) was used to quantitate in vivo production of IFN-{gamma} in mice undergoing GVHD. The IVCCA increases the sensitivity of detection of cytokines measured by a factor of ~100. Briefly, mice are injected i.v. with 10 µg of a biotin-labeled neutralizing mAb to IFN-{gamma}, which binds some, but not all, of the cytokine shortly after it is secreted. Mice are bled 1 day after biotin-mAb injection, and concentrations of biotin-mAb-cytokine complexes are measured by ELISA, using microtiter plate wells coated with a mAb to an epitope on the cytokine that is not blocked by the injected biotin-labeled mAb. Biotin-labeled mAb-cytokine complexes in serum samples or standards are detected with streptavidin-HRP, followed by a tetramethylbenzidine substrate solution that generates a luminescent compound when cleaved by HRP. Plates were read with a Fluoroskan Ascent Fluorometer (Labsystems).

Kidney histopathology

Kidney tissue was fixed in 10% buffered formalin and processed for routine paraffin embedding and histological sectioning. Three-micron-thick sections, stained with periodic acid-Schiff stains, were blindly scored by a renal pathologist (M. Haas). The severity of proliferative glomerulonephritis (GN) score was determined according to a previously described semiquantitative scoring system developed for a murine model of lupus nephritis (32).

Serological studies

Mice were bled at the times indicated, and sera were tested by ELISA for the presence of IgG Ab to ssDNA, as described (33).

Statistical analysis

Normally distributed data were analyzed by either unpaired Student’s t test (for single comparisons) or ANOVA (for multiple comparisons), as indicated.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Agonist CD40 mAb treatment of DBA->F1 mice converts lupus-like GVHD to acute GVHD

Agonist CD40 signaling in normal mice using FGK115 mAb induces both B cell-stimulatory and Th1-promoting activities (1). To determine whether one of these two properties prevails in the setting of an in vivo alloantigen-driven naive T cell response, we tested the ability of FGK115 to alter disease phenotype at 2 wk in the DBA->F1 model of lupus-like chronic GVHD based on previous work demonstrating that coadministration of agents that promote CD8 CTL activity in this P->F1 combination converts disease phenotype from chronic to acute GVHD (23, 34). DBA->F1 mice received 5 daily doses of FGK115 (100 µg i.v.) beginning at the time of donor cell transfer. Untreated DBA->F1 and B6->F1 mice served as positive controls for chronic and acute GVHD phenotypes, respectively. We have previously demonstrated that flow cytometric parameters at 2 wk after donor cell transfer can serve as early surrogate markers for longer-term clinical GVHD outcomes such as mortality and lupus-like renal disease in the P->F1 model (reviewed in Ref. 35). Surrogate markers include donor T cell engraftment, host B and T cell numbers, Fas and FasL up-regulation, and, to a lesser degree, serum autoantibody levels (22, 36, 37). Typical results for acute and chronic GVHD at 2 wk after parental cell transfer are shown in Fig. 1. Untreated DBA->F1 chronic GVHD mice exhibited the following: ~2-fold increase in total spleen cells and host B cells compared with uninjected F1 mice (Fig. 1A); engraftment of predominately donor CD4+ T cells (Fig. 1B); low level Fas and FasL expression on host B cells and donor T cells, respectively (Fig. 1C); and a 5-fold elevation of serum anti-ssDNA ab compared with normal F1 mice (Fig. 1D). In contrast, untreated B6->F1 acute GVHD mice exhibited the following: a 3-fold reduction in total splenocyte numbers compared with normal F1 mice and near complete elimination of host B cells (Fig. 1A); engraftment of both CD4+ and CD8+ donor T cells (Fig. 1B); significant up-regulation of Fas and FasL on host B cells and donor CD8+ T cells, respectively (Fig. 1C); and no significant elevation of serum anti-ssDNA ab compared with normal F1 mice (1D). Anti-CD40 mAb treatment of DBA->F1 chronic GVHD mice significantly altered the chronic GVHD phenotype and induced the following features typical of acute GVHD: 1) significant reduction of total splenocytes and near complete elimination of host B cells (Fig. 1A); 2) significant up-regulation of Fas on host B cells and FasL on donor T cells (Fig. 1C); and 3) significant reduction in serum anti-ssDNA Ab compared with untreated chronic GVHD mice (Fig. 1D). Taken together, these data support the idea that agonist CD40 stimulation converts chronic GVHD to acute GVHD in DBA->F1 mice. Of note, donor T cell engraftment in anti-CD40-treated DBA->F1 mice was reduced compared with acute GVHD mice (Fig. 1B), suggesting that donor T cell down-regulation may be accelerated with anti-CD40 mAb treatment.


Figure 1
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  		FIGURE 1. Early delivery of a CD40 signal converts GVHD phenotype from chronic to acute in DBA->F1 mice. Acute (B6->F1) and chronic (DBA->F1) GVHD were induced, as described in Material and Methods. DBA->F1 chronic GVHD received five daily doses of 100 µg of anti-CD40 mAb i.v. beginning at the time of donor cell transfer. After 2 wk, mice were sacrificed, and donor and host splenic lymphocyte subsets were analyzed by flow cytometry and serum was assessed for the presence of anti-ssDNA Ab, as described in Material and Methods. Values are shown as group mean ± SEM. Splenocytes and host B cells (A) and engrafted donor CD4+ and CD8+ T cells (B) are shown as total cell number x 10–6. C, Mean percentage Fas-positive host B cells and FasL-positive donor T cells. D, Mean serum anti-ssDNA Ab (n = 4–5 mice/group). Similar results were seen in an additional independent experiment. Anti-CD40-treated (aCD40), chronic GVHD (cGVHD) mice were compared with untreated acute GVHD (aGVHD) or chronic GVHD mice by Student’s t test. *, p < 0.05; ***, p < 0.001.

 
Follow-up experiments demonstrated that a single injection of anti-CD40 mAb given either i.p. or i.v. at day 0 or days 0 and 3 after donor cell transfer is as effective as five daily i.v. doses in inducing acute GVHD phenotype in DBA->F1 mice as measured by splenocyte reduction and host B cell elimination (Fig. 2A); increased donor CD8+ T cell engraftment (Fig. 2B); increased Fas and FasL up-regulation (Fig. 2C); and reduced serum autoantibodies (Fig. 2D). The ability of a single dose of anti-CD40 mAb to induce an acute GVHD phenotype at 2 wk was lost if administration was delayed until day 7 after donor cell transfer (data not shown). These results support the idea that CD40 signaling early (days 0–3) in DBA->F1 mice promotes anti-host CTL typical of acute GVHD.


Figure 2
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  		FIGURE 2. A single dose of anti-CD40 mAb early after donor cell transfer is as effective as multiple doses in inducing an acute GVHD phenotype in DBA->F1 mice. Chronic GVHD was induced, and mice were assessed at 14 days after donor cell transfer, as described for Fig. 1. Chronic GVHD (DBA->F1) mice were either untreated or received 100 µg of anti-CD40 mAb i.v. or i.p. as a single dose (day 0) or two doses (day 0, day 3). Values are shown as group means ± SEM (n = 3–5 mice/group). A, Total number (x10–6) of splenocytes and host B cells, and B, engrafted donor CD4+ and CD8+ T cells. C, Percentage positive Fas-expressing host B cells and FasL-expressing donor T cells; D, serum anti-ssDNA Ab (U/ml). *, p < 0.05; **, p < 0.01; ***, p < 0.001, for this figure and all subsequent figures.

 
Elimination of host B cells in anti-CD40 mAb-treated DBA->F1 mice is donor CD8+ T cell dependent

Host B cell elimination in B6->F1 acute GVHD mice is an in vivo correlate of donor anti-host CTL activity (38). The data in Fig. 1 strongly suggest that anti-CD40 mAb promotes host B cell elimination by inducing donor anti-host CTL. To confirm this idea and eliminate the possibility that anti-CD40 mAb directly depletes host B cells, anti-CD40 mAb was administered to F1 mice receiving donor CD4+, but not CD8+ T cells. Because the donor anti-host CTL response is stronger in B6 vs DBA donor T cells (22), we transferred B6 CD4+ parental T cells into BDF1 recipients (B6 CD4->F1). If anti-CD40 mAb promotes host B cell elimination by a CD8+ T cell-independent mechanism (e.g., direct depletion or induction of CD4+ CTL), F1 mice receiving donor CD4+ cells and anti-CD40 mAb should exhibit host B cell elimination at 2 wk rather than the expected B cell expansion. As shown in Fig. 3, mice receiving B6 CD4+ T cells alone or with control mAb exhibited a significant increase in total splenocytes (Fig. 3A), host B cell numbers (Fig. 3B), and serum anti-ssDNA ab (Fig. 3C) compared with normal F1 mice consistent with chronic GVHD, as previously described (22). B6 CD4->F1 mice receiving anti-CD40 mAb exhibited no significant reduction in any of these three parameters below values for either control F1 or control chronic GVHD mice. These results do not support the presence of donor CD4+ CTL or a direct B cell-depleting effect of anti-CD40 mAb, and instead indicate that donor CD8 T cells are absolutely required for the induction of the acute GVHD-like parameters seen in Figs. 1 and 2. Interestingly, F1 mice receiving anti-CD40 mAb without donor splenocytes also exhibited an increase in total splenocytes, in host B cells, and in anti-ssDNA comparable to control chronic GVHD mice consistent with the previously described B cell-stimulatory effect of anti-CD40 mAb (1). Similarly, the Th1-promoting properties of anti-CD40 mAb (1) are observed in this experiment as a striking up-regulation of Fas on host B cells in F1 mice receiving anti-CD40 mAb (with or without donor splenocytes) compared with F1 mice not receiving anti-CD40 mAb (Fig. 3D). Fas up-regulation in the P->F1 model is an IFN-{gamma}-dependent event (37) and supports the idea that IFN-{gamma} production is increased in DBA->F1 mice receiving anti-CD40 mAb. Thus, in the P->F1 model, CD40 stimulation results in both B cell stimulation and CMI/Th1-promoting properties; however, in the absence of donor CD8 T cells, the B cell-stimulatory properties prevail. These results support the conclusion that CD40 stimulation early in the course of DBA->F1 chronic GVHD promotes donor anti-host CD8, but not CD4, CTL activity, resulting in elimination of host B cells.


Figure 3
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  		FIGURE 3. Conversion of chronic GVHD to acute following anti-CD40 mAb treatment is CD8+ T cell dependent. Chronic GVHD was induced by transferring 12 x 106 B6 CD4+ (CD8+ T cell-depleted) splenocytes into BDF1 mice, as described in Material and Methods, and disease phenotype was assessed at day 14 after transfer. B6 CD4->F1 chronic GVHD mice were either untreated or received five daily doses of 100 µg of anti-CD40 mAb or control mAb i.v. starting at the time of donor cell transfer. Values are shown as group mean ± SEM (n = 4–5 mice/group) for A, total number of splenocytes; B, host B cells; C, serum anti-ssDNA mAb (U/ml); and D, Fas expression on host B cells (percent positive).

 
CD40 stimulation induces an accelerated acute GVHD phenotype in DBA->F1 mice

If anti-CD40 mAb promotes donor CD8 CTL and induces an acute GVHD in DBA->F1 mice, as suggested by the foregoing data, it is surprising that the number of donor CD8+ T cells engrafted at 2 wk is significantly less than that seen for B6->F1 acute GVHD mice (Fig. 1B). Donor CD8+ T cell engraftment in B6->F1 mice typically peaks at ~day 10 after donor cell transfer and then declines from days 10–14, reflecting CD8 CTL down-regulation (39). It is possible that the reduced donor T cell engraftment at day 14 seen in DBA->F1 mice receiving anti-CD40 mAb reflects accelerated donor CD8 CTL maturation and down-regulation compared with that of untreated B6->F1 acute GVHD mice (40). To test this possibility, the kinetics of donor T cell maturation and host B cell elimination were determined in DBA->F1 mice receiving a single dose of anti-CD40 mAb at day 0 and compared with either control mAb-treated DBA->F1 chronic GVHD or untreated B6->F1 acute GVHD mice. Compared with B6->F1 acute GVHD mice, anti-CD40 mAb treatment of DBA->F1 mice results in the following: 1) greater peak (day 5) numbers of donor CD4+ T cells (Fig. 4A); 2) an earlier peak in donor CD8+ T cells (Fig. 4B); and 3) a greater and earlier peak in host B cell expansion, followed by accelerated elimination (Fig. 4C). Regarding host T cells (Fig. 4, D and E), acute B6->F1 mice exhibit a peak in host CD8 T cells at day 10 (Fig. 4E), corresponding to the host anti-donor CD8 cytolytic response previously described by Kosmatopoulos et al. (41, 42). After day 10, both CD4 and CD8 host cells decline due to elimination by the stronger donor anti-host CD8 cytolytic response. By contrast, control DBA GVHD mice exhibit a gradual expansion of host T and B cells that persists out to day 14 due to defective donor anti-host CD8 CTL effector maturation and no elimination of host B cells (22). Anti-CD40 mAb treatment of DBA->F1 mice accelerates initial host CD4 and CD8 expansion as shown by a peak in host CD4 (Fig. 4D) and CD8 (Fig. 4E) T cells at day 5, several days earlier than that seen for either control DBA->F1 or B6->F1 mice and consistent with an accelerated host anti-donor response. Anti-CD40-treated DBA->F1 mice also exhibit an accelerated decline in host T cells (compared with B6->F1 mice) due to the accelerated donor anti-host response shown in Fig. 4, B and C.


Figure 4
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  		FIGURE 4. Anti-CD40 mAb treatment of DBA->F1 mice induces an accelerated acute GVHD phenotype. Acute (B6->F1) and chronic (DBA->F1) GVHD were induced, as described in Material and Methods. DBA->F1 chronic GVHD mice received a single 100 µg dose of anti-CD40 mAb or control mAb i.v. at the time of GVHD induction. B6->F1 mice received no mAb. An additional control was normal F1 mice receiving a single dose of anti-CD40 mAb and no donor splenocytes. Splenocytes were analyzed by flow cytometry on days 2, 3, 4, 5, 7, 10, and 14 after parental cell transfer for donor T cell engraftment, host B cells and T cells, and host DC- and Fas-positive host B cells, as described in Material and Methods. Mice were bled at the indicated time points, and serum IFN-{gamma} was determined by IVCCA. Each time point represents a separate independent experiment. Sequential kinetics are shown for the following: A, donor CD4+ T cells; B, donor CD8+ T cells; C, host B cells; D, host CD4 T cells; E, host CD8 T cells; F, CD11c+ host DC; G, serum IFN-{gamma}; and H, the percentage of Fas-positive host B cells. Shown in I are representative dot plots from individual mice from day 5 and day 10 experiments for donor T cell engraftment and host B cells, respectively. Shown in J are representative tracings of Fas up-regulation on host B cells for days 10 and 14. Results are shown as group mean ± SEM (n = 3–5 mice/group). ***, p < 0.001. Chronic GVHD plus anti-CD40 vs acute GVHD.

 
Anti-CD40 mAb also induces an earlier peak in host dendritic cell (DC) expansion (Fig. 4F), earlier peak in serum IFN-{gamma} levels (Fig. 4G), and earlier up-regulation of Fas on host B cells (Fig. 4H) and FasL on donor CD8 T cells (data not shown). As shown previously (33, 37), acute GVHD is characterized by significantly greater IFN-{gamma} production than is chronic GVHD, which in turn results in greater up-regulation of Fas on host B cells and FasL on donor CD8 T cells. Thus, anti-CD40 mAb treatment of DBA->F1 mice induces not only a stronger serum IFN-{gamma} production characteristic of acute GVHD, but the IFN-{gamma} response is accelerated compared with acute GVHD, which in turn accelerates Fas/FasL up-regulation, CTL effector function, elimination of host T and B cells, and down-regulation of donor CD8 T cells compared with acute GVHD. Taken together, these results demonstrate that agonist anti-CD40 mAb administration not only has immune modulatory properties in that it can convert 2-wk disease phenotype in DBA->F1 mice from chronic lupus-like GVHD to CTL-mediated acute GVHD, but it also can accelerate development of that phenotype. Interestingly, the initial B cell expansion in anti-CD40-treated DBA–->F1 mice is donor T cell independent and comparable to host B cell expansion seen in anti-CD40-treated normal F1 mice (Fig. 4C, days 2–5). In contrast, expansion of host DC in anti-CD40-treated DBA->F1 mice is donor T cell dependent and is not seen in anti-CD40-treated F1 mice (Fig. 4F, days 2–5). Representative two-color flow cytometry dot plots from individual mice are shown in Fig. 4I for donor and host CD4 and CD8 T cells (day 5) and host B cells (day 10) for the following: F1 mice receiving anti-CD40; DBA->F1 plus control mAb; DBA->F1 plus anti-CD40; and B6->F1 acute GVHD mice. These results confirm that anti-CD40 mAb accelerates the engraftment of donor CD4 and CD8 T cells and the elimination of host B cells in DBA->F1 mice, as shown by an increase in the absolute numbers (rather than the percentage) of cells. Individual single-color tracings demonstrating up-regulation of Fas on gated host B cells are shown in Fig. 4J for the following: normal F1; DBA->F1 plus control mAb; DBA->F1 plus anti-CD40; and B6->F1 acute GVHD mice at both days 10 and 14, and demonstrate that Fas up-regulation is accelerated in anti-CD40-treated DBA->F1 mice as shown by greater up-regulation at day 10 compared with acute GVHD mice.

Anti CD40 treatment of DBA->F1 mice induces in vivo cytolytic activity

We have previously demonstrated in acute GVHD that host B cell reduction is a more sensitive indicator, albeit indirect, of anti-host CTL activity than is direct measurement of in vitro cytolytic activity (e.g., 51Cr release) even without an in vitro sensitization period (i.e., direct ex vivo killing) (38). Recently, it has become feasible to directly test in vivo killing using CFSE-loaded target cells (29, 43). To determine whether accelerated host B cell reduction is due to accelerated in vivo anti-host CTL effector function in anti-CD40-treated DBA->F1 mice, we measured in vivo anti-host CTL activity at day 7 using CFSE-loaded target cells of both B6 and DBA parental origin. We chose day 7 based on the results in Fig. 4, demonstrating a decline in host B cells at this time for anti-CD40-treated DBA->F1 mice (consistent with active anti-host cytolytic activity) that is well before the decline in host B cells in B6->F1 acute GVHD mice (day 10). In DBA->F1 mice, anti-host cytolytic activity is measured as killing on B6 targets, whereas in B6->F1 mice, anti-host activity is measured as killing of DBA targets. The reverse is true for measurement of F1 anti-parent killing. When cytolytic activity is calculated as anti-host killing relative to anti-donor killing (Fig. 5A), control F1 mice receiving anti-CD40 exhibit low level killing (mean value <10%). Anti-host killing in control DBA->F1 chronic GVHD (with or without control mAb) and in B6->F1 acute GVHD mice was significantly elevated compared with F1 plus anti-CD40 mAb controls and ranged from 20 to 40%. Anti-CD40 mAb treatment of DBA->F1 mice resulted in striking elevations in anti-host killing that was significantly greater than all controls, including control acute and chronic GVHD mice. These general trends are confirmed when killing is shown as the absolute number of surviving B6 (Fig. 5B) or DBA (Fig. 5C) target cells. Anti-CD40 treatment of normal F1 mice results in a small amount of nonspecific killing that is not significant for either target. Control DBA->F1 mice exhibit a significant reduction in host (B6) targets compared with untreated or anti-CD40-treated F1 mice. Anti-CD40 mAb treatment of DBA->F1 mice results in significantly greater killing of host targets compared with control DBA->F1 mice. The significant reduction of B6 targets in B6->F1 mice (Fig. 5B) represents F1 anti-parent killing. Anti-host killing is shown for B6->F1 mice in Fig. 5C as a significant reduction in DBA targets compared with either untreated or anti-CD40-treated F1 mice. Interestingly, no significant F1 anti-parent killing activity is demonstrable for control mAb-treated DBA->F1 mice compared with F1 controls (Fig. 5C); however, anti-CD40 mAb-treated mice exhibit significant reductions in DBA targets (Fig. 5C) indicative of anti-CD40-mediated enhanced F1 anti-parent cytotoxicity that coincides with the increase in host CD8 T cells seen in Fig. 4E. Representative flow cytometry curves are shown in Fig. 5D and demonstrate striking reductions in B6 (CFSElow) host targets in anti-CD40 mAb-treated DBA->F1 mice. Acute GVHD mice exhibit significant reduction of DBA (CFSEhigh) host targets. Taken together, these results indicate that the major effect of anti-CD40 mAb treatment of DBA->F1 mice is to promote Ag-specific in vivo cytotoxicity to include both donor anti-host and host anti-donor killing. The strong anti-host killing at day 7 of disease is consistent with an accelerated form of acute GVHD that overcomes the anti-CD40-enhanced host anti-donor response. The significantly greater number of host B cells at day 5 with anti-CD40 treatment and their near complete elimination by day 10 further support an enhanced in vivo anti-host CTL response.


Figure 5
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  		FIGURE 5. Anti-CD40 mAb induces accelerated anti-host CTL effector function in DBA->F1 mice. GVHD induction and experimental groups are as outlined in Fig. 4, with the addition of untreated chronic DBA->F1 GVHD as an extra control group. At day 7 after donor cell transfer, mice were injected i.v. with a 1:1 ratio of CFSElow B6 targets and CFSEhigh DBA targets, as described in Materials and Methods. After 5 h, the percentage of surviving B6 and DBA targets was determined by flow cytometry. The percentage of anti-host killing relative to parental cells was determined, as described in Materials and Methods, and is shown in A. The actual number of surviving targets was calculated as described in Materials and Methods, and is shown in B for B6 targets and in C for DBA targets. Symbols represent values for individual mice. Shown in D are representative histograms from each group of gated CFSElow/high target cells and their relative percentage.

 
Anti-CD40 mAb accelerates initial donor CD8 T cell, but not CD4 T cell activation

As shown in Fig. 4, A and B, anti-CD40 mAb treatment induces significantly greater donor T cell engraftment by day 5 compared with control mAb-treated DBA->F1 chronic GVHD or untreated B6->F1 acute GVHD mice. To determine whether this effect is due to acceleration of initial donor CD4 and/or donor CD8 T cell activation, CFSE-loaded donor T cells were injected into BDF1 mice, and the kinetics of activation was assessed. Comparing anti-CD40 mAb treatment with control mAb treatment in DBA->F1 mice at days 2, 3, and 4 (Fig. 6), there was a small, but significant enhancement of the average number of divisions per cell (Fig. 6A) and the percentage (Fig. 6C) of donor CD4+ T cells undergoing ≥1 division at day 2; however, there were no significant differences at days 3 and 4 for either parameter. In contrast, donor CD8+ T cells in mice receiving anti-CD40 mAb exhibited significantly greater average number of divisions per cell (Fig. 6B) at day 4 and a significant increase in the percentage of cells undergoing at least one division (Fig. 6D) at days 3 and 4 after cell transfer compared with control mAb-treated DBA->F1 mice. These results are consistent with accelerated initial activation of primarily donor CD8 T cells with CD40 stimulation. Representative tracings from individual mice at day 4 are shown in Fig. 6E and demonstrate comparable expansion of donor CD4 T cells, but more rapid expansion (greater percentage of cells undergoing at least one division) for donor CD8 T cells from anti-CD40-treated mice.


Figure 6
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  		FIGURE 6. Anti-CD40 treatment accelerates initial donor CD8+ T cell activation. DBA->F1 chronic GVHD was induced using CFSE-labeled DBA donor splenocytes, as described in Material and Methods. GVHD mice received 100 µg of either anti-CD40 mAb or control mAb i.v. at the time of donor cell transfer, and splenocytes were analyzed by flow cytometry on days 2, 3, and 4 after parental cell transfer. Results are shown as group mean ± SEM for average number of divisions per cell for donor CD4+ (A) or CD8+ (B) T cells and for the percentage of donor CD4+ (C) and CD8+ (D) cells undergoing at least one division (n = 3–5 mice/group). *, p < 0.05 anti-CD40 vs control Ab; **, p < 0.01 anti-CD40 vs control Ab. Shown in E are representative histograms for day 4 and the relative percentage of cells that have undergone at least one division (left) or are undivided (right).

 
CD40 stimulation accelerates DC and B cell activation in DBA->F1 mice

The data in Fig. 6 raise the possibility that the enhancement of donor CD8 CTL seen with CD40 stimulation may have a CD4-independent component, possibly mediated by accelerated expansion of host DC seen in Fig. 4F. To test this possibility, we examined B cell and DC activation markers at day 7 after donor cell transfer. As shown in Fig. 4, anti-CD40-treated DBA->F1 mice exhibit a significant increase in host B cells (Fig. 4C) and DC (Fig. 4F) compared with control mAb-treated DBA->F1 mice. This increase in cell numbers was associated with increased activation, as shown by greater up-regulation of MHC II on host B cells (Fig. 7A) and DC (Fig. 7B) and a greater percentage of host B cells (Fig. 7C) and DC (Fig. 7D) expressing the costimulatory molecule CD80 compared with either control-treated DBA->F1 mice or CD40 mAb-treated F1 mice. Of note, anti-CD40 mAb-induced host B cell expansion was comparable in mice receiving donor cells or no donor cells (Fig. 4C), although in the absence of donor cells expanded host B cells exhibited no significant increase in MHC II and a small, but significant increase in CD80 expression (p < 0.001). Thus, anti-CD40 mAb has a direct B cell-stimulatory effect independent of donor T cells that boosts B cell numbers, but does not result in sustained B cell activation in the absence of Ag-activated (i.e., donor) T cells. In contrast, the striking expansion of host DC numbers required both anti-CD40 mAb and donor cells (Fig. 4F) and is associated with significant greater up-regulation of MHC II and especially CD80 compared with control mAb DBA->F1 mice or in anti-CD40 F1 mice not receiving DBA donor cells. These results indicate that accelerated up-regulation of activation markers (MHC II and CD80) on host DC and B cells requires both donor T cell activation and CD40 stimulation.


Figure 7
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  		FIGURE 7. CD40 administration accelerates DC and B cell activation in DBA->F1 mice. Experimental groups are as described for Fig. 4. DBA->F1 chronic GVHD mice received ether 100 µg of anti-CD40 mAb or control mAb i.v. at the time of GVHD induction. Acute B6->F1 mice received no mAb. Additional controls consisted of normal BDF1 mice receiving 100 µg of anti-CD40 mAb at day 0 or no mAb. Splenocytes were analyzed by flow cytometry at day 7 after parental cell transfer, and MHC class II expression is shown as mean channel fluorescence for host B cells (A) and host DC (B). CD80 expression is shown as the percent positive cells for host B cells (C) and host DC (D). Values represent group mean ± SEM (n = 4–5 mice/group).

 
To determine whether the anti-CD40-induced accelerated expansion (Fig. 4, C and F) and activation (Fig. 7) of host B cells and DCs are associated with skewing toward a particular subset, we analyzed DC subsets at days 5, 10, and 14 after donor cell transfer. Although all DC (CD11c+) subsets are increased in anti-CD40 mAb-treated DBA->F1 mice at day 5 (Fig. 8), the most striking increase is for the CD4+CD8– DC (Fig. 8A), which was ~10-fold greater in anti-CD40-treated DBA->F1 chronic GVHD mice than in either control mAb-treated DBA->F1 or untreated acute B6->F1 GVHD mice. A small, but significant increase was also seen in anti-CD40-treated DBA->F1 mice at day 5 for CD4–CD8+ (Fig. 8B) and CD4–CD8– (Fig. 8C) subsets. At day 10, B6->F1 acute GVHD mice exhibit a significant increase in both the CD4–CD8+ (Fig. 8B) and CD4–CD8– DC subsets (Fig. 8C), but not in CD4+CD8– DC (Fig. 8A), whereas control DBA->F1 chronic GVHD mice exhibit little expansion of any DC subsets over this same time. By day 14, most host DC have been killed by donor anti-host CTL in acute GVHD mice and in anti-CD40 mAb-treated DBA->F1 mice.


Figure 8
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  		FIGURE 8. Anti-CD40 mAb induces early DC expansion in DBA->F1 mice, particularly the CD4+CD8– subset. Experimental groups and protocol are as described for Fig. 4. Splenocytes were analyzed by flow cytometry on days 5, 10, and 14 after parental cell transfer. CD11chigh DC subsets were analyzed based on their expression of CD4 and CD8 markers. Results shown are the group mean ± SEM x 10–6 (n = 4–5 mice/group) for CD4+CD8– DC (A); CD4–CD8+ DC (B); and CD4– CD8– DC (C). Values of p compare anti-CD40 mAb-treated DBA->F1 with control mAb-treated DBA->F1 mice.

 
The kinetics of B cell subset expansion are shown in Fig. 9 for germinal center, marginal zone, transitional-1 (T1), and transitional-2 (T2) B cell subsets. As shown in Fig. 4, nearly all host B cells have been eliminated by day 10 in anti-CD40 mAb-treated DBA->F1 mice and by day 14 in acute B6->F1 mice. In control mAb-treated DBA->F1 mice, host B cells are not eliminated and expand from days 5 to 14. Thus, the effect of anti-CD40 mAb is best seen in Fig. 9 at day 5 and demonstrates that all four B cell subsets are strikingly increased compared with acute or chronic GVHD control mice without evidence of major subset skewing. Anti-CD40 mAb treatment of normal F1 mice not receiving DBA donor cells results in a similar increase in B cells for all subsets except the T1 subset, indicating that, in general, anti-CD40-induced B cell expansion is donor T cell independent. In acute B6->F1 mice, initial expansion of host B cells before CTL elimination is primarily comprised of T1 B cells, whereas the sustained B cell expansion in control DBA->F1 chronic GVHD mice is comprised primarily of follicular and T1 B cells.


Figure 9
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  		FIGURE 9. Anti-CD40 mAb expands all four B cell subsets. Experimental groups and protocol are as described for Fig. 4. Splenocytes were analyzed by flow cytometry on days 5, 10, and 14 after parental cell transfer. Gated host B220+ B cells (MHC IIb positive for DBA->F1 mice and MHC IId positive for B6->F1 mice) were further analyzed based on their expression of CD21/CD35 and CD23 markers and shown as A, marginal zone (CD21/CD35high, CD23low); B, follicular B cells (CD21/CD35int, CD23high); C, type 1 (T1) transitional B cells (CD21/CD35neg, CD23neg); and D, type 2 (T2) transitional B cells (CD21/CD35+, CD23+). Results shown are the group mean ± SEM (n = 4–5 mice/group). Values of p compare anti-CD40 mAb-treated DBA->F1 with control mAb-treated DBA->F1 mice.

 
CD40 stimulation bypasses the requirement for CD4 T cell help in donor CD8 CTL effector generation

The demonstration that CD40 stimulation accelerates activation of host DC and B cell (Figs. 7 and 8) and accelerates initial donor CD8 activation, but not donor CD4 activation (Fig. 6), raises the possibility that CD40 stimulation bypasses the requirement for donor CD4+ T cell help for effector donor CD8 CTL maturation. To address this possibility, BDF1 mice were injected with purified DBA CD8+ donor T cells with and without anti-CD40 mAb. In two separate experiments, we were unable to detect donor CD8 T cell engraftment at either day 7 or day 14 in any group receiving donor cells. To eliminate the possibility that host NK cells reject donor CD8 T cells before they could become engrafted, recipient mice were pretreated with anti-NK1.1 mAb before donor cell transfer. As shown in Fig. 10A, pretreatment with anti-NK1.1 resulted in low level engraftment of DBA donor CD8+ T cells in F1 mice receiving control mAb and donor CD8 T cells, whereas a significant increase in donor CD8+ T cell engraftment was seen both at days 7 (~5-fold) and 14 (3-fold) in F1 mice receiving anti-CD40 mAb and donor CD8+ T cells. The donor cell inocula contained <1.0% CD4+ T cells and, as shown in Fig. 10B, there was no detectable donor CD4 T cell engraftment over uninjected control F1 mice at either time for any group. The B cell-stimulatory effect of anti-CD40 mAb is seen in Fig. 10C as a significant increase in host B cells (~3-fold) at day 7 in F1 mice both with or without accompanying donor CD8+ T cells. Importantly, host B cells are completely eliminated by day 14 in mice receiving anti-CD40 mAb and donor CD8+ T cells, whereas B cells decline to near normal levels and are not eliminated in mice receiving anti-CD40 mAb in the absence of donor CD8+ T cells (Fig. 10C). Serum IFN-{gamma} at day 7 was not significantly increased over control in the absence of donor CD4+ T cells (data not shown); however, in the absence of transferred donor CD4 T cells, day 7 may no longer be the time of peak values. Up-regulation of Fas on host B cells is IFN-{gamma} dependent in this model (37), and both anti-CD40 mAb treatment of control F1 and CD8 ->F1 mice significantly increases Fas up-regulation over mice not receiving anti-CD40 mAb, with the highest levels seen in mice receiving both anti-CD40 mAb and donor CD8 T cells (Fig. 10D). The striking increase in host DC seen with anti-CD40 mAb in DBA->F1 mice in Fig. 4F when both donor CD4 and CD8 T cells are transferred is also seen in mice receiving anti-CD40 mAb and donor CD8 T cells only (Fig. 10E), indicating that anti-CD40 mAb-induced DC expansion requires Ag-specific T cell activation and is CD4 independent, CD8 dependent. Lastly, in vivo anti-host CTL activity was assessed using CFSE-loaded targets. As shown in Fig. 10F, strikingly elevated anti-host CTL activity was seen for F1 mice receiving donor CD8 T cells and anti-CD40, whereas no detectable anti-host killing over control mice was observed in F1 mice receiving donor CD8 T cells and control mAb. These results demonstrate that anti-CD40 mAb promotes CD8 T cell expansion and CTL effector generation in a CD4-independent manner, consistent with licensing of DC.


Figure 10
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  		FIGURE 10. Anti-CD40 mAb bypasses the requirement for donor CD4 T cell help in the generation of donor CD8 CTL and acute GVHD. Normal F1 mice were treated with 200 µg of anti-NK 1.1 mAb i.p. 2 days prior to donor cell transfer and on day 3 after transfer. Donor CD8+ T cells were purified, as described in Material and Methods. The 107 purified CD8+ T cells were transferred into anti-NK 1.1-treated normal F1 mice that also received either 100 µg of anti-CD40 mAb or control mAb i.v. at the time of transfer. Other controls consisted of anti-NK 1.1-treated normal F1 mice that received no additional mAb or received 100 µg of anti-CD40 mAb i.v on day 0. Two separate experiments were performed in which mice were tested at either day 7 or 14 and assessed by flow cytometry for the numbers of A, donor CD8 T cells; B, donor CD4 T cells; or C, host B cells; D, the percentage of Fas-positive host B cells; and E, host DC (CD11c+). Results are shown as group mean ± SEM (n = 3). F, In an additional experiment using the same experimental protocol, in vivo cytolytic activity was determined using CFSE-labeled B6 or DBA strain targets, and the relative percentage killing of host targets was calculated as described in Materials and Methods. Values are shown for individual mice.

 
A single dose of anti-CD40 at the time of donor cell transfer prevents lupus-like renal disease in DBA->F1 mice

Long-term studies were performed to determine whether the elimination of host B cells seen at 2 wk in anti-CD40 mAb-treated DBA->F1 chronic GVHD mice was predictive of longer-term improvement of lupus-like features, as measured by serum anti-ssDNA Ab, lymphocyte subsets, and renal disease. It should be noted that because the dose of 80 x 106 DBA donor cells is just above the threshold for induction of chronic GVHD (44, 45), it is an optimal dose for detecting agents that promote donor CD8 CTL activity. Conversely, it is a relatively low dose for inducing lupus-like renal disease, which is better seen at doses of >107 DBA donor cells (44). The purpose of this study was to determine the long-term effects of the experimental conditions that result in CTL promotion at 2 wk (i.e., a single dose of anti-CD40 mAb and a dose of 80 x 106 DBA donor cells); therefore, a mild renal disease in control DBA->F1 mice is expected. As shown in Fig. 11A, control mAb-treated DBA->F1 mice exhibit an initial rise in serum anti-ssDNA Ab that peaks at week 4 and remains elevated out to week 12. By contrast, a single dose of anti-CD40 mAb given at the time of donor cell transfer (day 0) blocks the week 4 peak seen in control DBA->F1 mice and results in anti-ssDNA Ab levels that are significantly reduced at both the 4- and 6-wk time points. Interestingly, by week 8, anti-CD40-treated DBA->F1 mice begin to exhibit serological escape, as evidenced by a gradual rise in serum anti-ssDNA Ab levels and wide SE bars due to large individual variation. For example, at 4 wk, five of five mice in the anti-CD40-treated DBA->F1 group exhibited anti-ssDNA Ab levels that did not differ significantly from uninjected F1 controls with values well below 50 U. Using 50 U as an arbitrary indicator of significantly elevated anti-ssDNA levels, anti-CD40-treated DBA->F1 mice exhibited the following trend: at 8 wk, one of five mice were positive (≥50 U); at 10 wk, two of five mice were positive; at 12 wk, four of five mice were positive. Analysis of lymphocyte subsets at week 12 (Fig. 11, B–D) demonstrated that residual host B cells in anti-CD40-treated DBA->F1 mice were significantly lower than in control mAb DBA->F1 chronic GVHD (Fig. 11B), as expected based on their near complete elimination seen at week 2 (Fig. 4C); however, host B cells were significantly greater than that seen for acute GVHD mice, indicating that despite the near complete elimination of splenic host B cells in anti-CD40-treated DBA->F1 mice seen at 2 wk (Fig. 4C), B cell reduction was not maintained long-term to the degree seen in acute GVHD mice. Repopulation by donor B cells was equivalent in both acute GVHD and anti-CD40-treated chronic GVHD mice (p = NS). Acute GVHD mice also exhibited significantly greater residual donor CD4 and CD8 engraftment (Fig. 11C) and greater elimination of host CD4 T cells (Fig. 11D) compared with anti-CD40-treated chronic GVHD mice. The rise in serum anti-ssDNA Ab in anti-CD40-treated chronic GVHD mice after week 8 most likely represents donor CD4 T cell collaboration with residual host B cells, as shown by the greater number of residual host B cells and reduced numbers of donor CD8 T cells in this group compared with control acute GVHD.


Figure 11
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  		FIGURE 11. Anti-CD40 mAb treatment of DBA->F1 chronic GVHD mice prevents lupus-like renal disease at 2 mo. Experimental and control groups and dosing of control mAb or anti-CD40 mAb are as described in Fig. 4. Mice were bled for anti-ssDNA at 2-wk intervals, and the results are shown in A. At 12 wk, mice were sacrificed. Analysis of splenic lymphocyte subsets by flow cytometry was performed and shown for host and donor B cells (B), and CD4 and CD8 T cells of donor (C) and host (D) origin. Values represent group mean ± SEM (n = 4–5 mice/group). E, Representative kidneys are shown from an individual mouse from each of the indicated groups (PAS stain, original magnification, x400). Normal control F1 (left panel) showed normocellular glomeruli without segmental lesions. Chronic DBA->F1 GVHD receiving anti-CD40 mAb and acute B6->F1 GVHD mice (right two panels) generally showed mild mesangial hypercellularity without segmental glomerular lesions. Fifty percent of the chronic GVHD mice not receiving anti-CD40 mAb showed glomerular crescent formation (arrow, second from left panel). A portion of a second glomerulus with a crescent, but not showing the glomerular tuft in the plane of the section, is indicated by an asterisk.

 
Despite the late rise in serum anti-ssDNA Ab, the incidence of lupus-like renal disease at 12 wk was completely blocked in DBA->F1 mice receiving anti-CD40 compared with control mAb (representative renal histology is shown in Fig. 11E). Specifically, no evidence of lupus-like renal disease was seen in controls (untreated and anti-CD40-treated F1 mice), and glomerular scores were uniformly <1. In contrast, lupus-like glomerular disease was observed in 5 of 10 control DBA->F1 mice (2 of 5 control mAb-treated and 3 of 5 untreated DBA->F1 mice), exhibiting a GN score of >2 that in each case was accompanied by glomerular changes such as crescent formation, hyalinization, and glomerular sclerosis (Fig. 11E). By contrast, four of five anti-CD40-treated DBA->F1 mice exhibited a GN score of ≤1, and no mice in this group exhibited crescent formation, glomerular sclerosis, or hyalinization (Fig. 11E). These results are similar to that seen in acute GVHD, 4 of 5 of which had GN scores of ≤1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study addresses the ability of CD40 stimulation to promote CTL in the setting of ongoing B cell activation and humoral autoimmunity. Using the P->F1 model of GVHD, we asked whether the CTL-promoting effects of an agonist anti-CD40 mAb could limit autoreactive B cell expansion seen in chronic GVHD and induce acute GVHD or would these effects be outweighed by the B cell-stimulatory properties of anti-CD40 mAb (46) and exacerbate lupus-like chronic GVHD. Because CD40:154 interactions are central to both normal B cell activation and to B cell hyperactivity in lupus (1, 7), it was possible that CD40 stimulation might not only exacerbate B cell hyperactivity in chronic GVHD mice, but also skew the immune milieu such that CTL responses were inhibited. Our results demonstrate that during the early stages of lupus induction, administration of agonist CD40 mAb does in fact promote B cell expansion in chronic GVHD mice; however, this trend is countered by CD40-induced accelerated CTL effector maturation, which then eliminates activated B cells. Specifically, compared with donor anti-host CTL kinetics in acute (B6->F1) GVHD mice, anti-CD40 mAb-treated lupus-like chronic (DBA->F1) GVHD mice exhibited earlier peaks in donor CD8 T cell engraftment, in Fas/FasL up-regulation, and in serum IFN-{gamma}, culminating in earlier elimination of host B cells by donor anti-host CTL effectors.

CD40 stimulation in our studies accelerated CD8 CTL effector maturation by bypassing the requirement for CD4 help, confirming previous work (47, 48, 49). This was directly demonstrated by the ability of purified donor CD8 T cells (in the absence of donor CD4 T cells) to induce acute GVHD only if F1 mice also received anti-CD40 mAb and not control mAb. This conclusion is also supported indirectly by our demonstration that CD40 stimulation induced earlier expansion and earlier activation of host DC. Interestingly, the ability of anti-CD40 mAb to increase the numbers of activated host DC was not seen in anti-CD40-treated normal F1 mice, indicating that anti-CD40-induced expansion of activated DC requires Ag-activated (donor) CD8 T cells (Fig. 10E). All DC subsets in anti-CD40-treated DBA->F1 mice were increased compared with untreated DBA->F1 mice; however, there was a striking increase at day 5 in the CD4+CD8– DC subset, which is a poor producer of IFN-{gamma} and IL-12p70 and promotes Th2 responses (50, 51, 52). The increase in this subset parallels the early B cell-stimulatory effects of CD40 mAb. In contrast, the CD4–CD8+ DC subset that promotes Th1 responses (50, 51, 52) was slightly, but significantly increased at day 5 in anti-CD40-treated DBA->F1 mice compared with untreated GVHD mice and followed a similar kinetic pattern as control acute GVHD mice and increasing out to day 10. Double-negative CD4–CD8– DC also increased by day 10 in acute GVHD mice, but not in anti-CD40-treated DBA->F1. Although anti-CD40-treated DBA->F1 mice mimicked other parameters of acute GVHD phenotype, the discrepancy in the numbers of double-negative DC is not understood at present.

Anti-CD40 mAb treatment of normal mice induced very low level cytolytic activity toward both parental targets that was not significantly different from untreated F1 mice. In contrast, anti-CD40 mAb strongly promoted ongoing Ag-specific cytolytic effector function to include donor anti-host and, to a lesser extent, host anti-donor killing. It has been previously shown that CTL-promoting agent such as rIL-12 will also convert chronic GVHD to acute GVHD in the DBA->F1 model by inducing DBA CD8 T cells to mature into effector CTL (23). Thus, promotion of CTL effectors, whether by agonist CD40 stimulation or by direct IL-12 administration, is one final common pathway by which chronic GVHD can be converted to acute GVHD in this model. IL-12 can also be induced by CD40 stimulation (53) and could contribute to the ability of anti-CD40 mAb to promote CTL effector function in addition to licensing DC and bypassing the need for CD4 T cell help.

In contrast to the T cell-dependent nature of anti-CD40-mediated DC expansion, anti-CD40-mediated B cell expansion was for the most part T cell independent. All four B cell subsets examined were expanded with anti-CD40 stimulation in DBA->F1 mice, and anti-CD40 treatment of normal F1 mice resulted in an expansion of all B cell subsets, except T1 subset. Interestingly, the T1 B cells were increased in both control acute and chronic GVHD. In chronic GVHD mice, early B cell hyperactivity is comprised primarily of the T1 and follicular B cell subsets.

Engagement of CD40 on B cells by T cell-expressed CD154 results in germinal center formation, clonal expansion, Ig secretion, and enhanced APC function of normal B cells. In lupus, T cell expression of CD154 is prolonged (5, 6), and excessive CD40:CD154 interactions are thought to contribute to B cell hyperactivity and pathogenic autoantibody production (7). Therapeutic CD40:CD154 blockade has been beneficial in both murine and human lupus; however, thrombotic complications have limited this approach in humans (8, 54). Nevertheless, B cells remain a therapeutic target in lupus, and newer approaches aimed at global B cell depletion such as the anti-CD20 mAb (Rituximab) appear promising (55). Side effects such as increased infections do not appear to be a major problem in nonimmunocompromised patients, with the exception of hepatitis B reactivation (56, 57). Nevertheless, Rituximab prevents both primary and secondary Ab responses in vivo to neo Ags (58), raising concerns about its long-term use.

A more targeted therapeutic approach in lupus in which only the pathogenic autoreactive B cells are eliminated is clearly desirable. Such an approach could be achieved by promoting CTL elimination of autoreactive B cells in an Ag-specific manner. Published reports support the idea that CTL may be an important, but understudied down-regulatory mechanism in lupus with the potential to limit autoreactive B cell expansion. Defective CTL function has been reported in both human and murine lupus (59); however, it is not clear whether this represents a primary predisposing defect, a secondary effect due to altered immunoregulation and defective IL-2 production characteristic of lupus (60, 61), or both. Supporting a primary role for CD8 CTL down-regulation are studies in murine lupus demonstrating that an intrinsic defect in pfp-mediated CTL effector function exacerbates disease expression. Using a model of spontaneous lupus-like disease, Peng et al. (16) demonstrated that pfp-deficient (pfp–/–) MRL/lpr mice exhibit accelerated end organ disease compared with pfp-intact MRL/lpr mice, indicating that pfp plays a regulatory role in limiting lupus expression. In an induced model of lupus-like disease, the P->F1 model of GVHD, the transfer of donor cells defective in CTL effector function induces a lupus-like chronic GVHD in mice that would otherwise develop acute GVHD. The effector defects that result in lupus-like GVHD may be either qualitative, e.g., pfp–/– donor cells (18); quantitative, e.g., reduced pCTL frequency (62); or a combination of both, as seen with DBA donor cells (21, 22, 36). The severity of the CTL defect in the donor inoculum correlates with the timing of lupus-like disease onset such that donor cells with severe defects in CTL effector generation (e.g., complete absence of CD8+ T cells or the use of DBA donor cells) induce B cell expansion by 14 days after donor cell transfer, whereas donor cells with less severe defects (pfp–/– or reduced pCTL frequency) do not exhibit B cell expansion until ≥8 wk.

Taken together, the above studies support the idea that not only can lupus result from a failure of endogenous CTL to control hyperactive B cells, but also that promotion of CTL specific for autoreactive B cells might be beneficial in lupus patients. This idea is supported by work of by Fan and Singh (63) in spontaneous NZB/W murine lupus. Using an Ag-specific strategy, it was demonstrated that therapeutic induction of CD8 CTL specific for pathogenic peptides was both feasible and beneficial. In human lupus, the antigenic specificity of the autoreactive T and B cells is not completely known and may differ among patients. For this reason, we have pursued an Ag-independent strategy. In the GVHD model, donor CD4 T cells provide help (signal 2) to all host B cells. Only those B cells that also encounter Ag through their BCR (signal 1) will become mature IgG-secreting B cells (20). In the absence of exogenous Ag administration, GVHD mice make IgG Ab to readily encounter self-Ag (e.g., anti-ssDNA) (64). Although the CTL generated in acute GVHD target all host B cells rather than only the autoreactive ones (37), it is the elimination of activated autoreactive host B cells that prevents lupus-like disease. A single dose of anti-CD40 mAb given at disease induction prevented lupus-like renal disease in DBA->F1 mice at 12 wk; however, a single dose may not be sufficient to control disease for longer periods, as shown by a rise in serum autoantibodies and in host B cell numbers at 12 wk compared with acute B6->F1 mice. For example, compared with anti-CD40-treated DBA->F1 mice, B6->F1 acute GVHD mice exhibit significantly greater numbers of donor CD4 and CD8 T cells and significantly lower numbers of host B cells at 12 wk, and exhibit no significant increase in serum anti-ssDNA Ab above control. In contrast, the reduced numbers of donor CD8 T cells and greater numbers of host B cells in anti-CD40-treated DBA->F1 mice are associated with a rise in serum anti-ssDNA Ab beginning as early as 8 wk, and most likely reflect impaired elimination of autoreactive host B cells. Reduced CD8 CTL numbers in anti-CD40-treated DBA->F1 mice could arise from defective donor CD4 T cell help (as suggested by the reduced numbers compared with B6->F1 mice at 12 wk) or result from defective memory CD8 CTL function as a consequence of initial CD40 mAb treatment that bypasses the need for CD4 T cell help. The development of memory CD8 CTL has been shown to be defective in the absence of CD4 T cell help during the initial activation of naive CD8 precursors (65, 66, 67, 68). Alternatively, these results may simply reflect the need for repeated mAb administration as seen with other nonspecific B cell-depleting agents (e.g., Rituximab) (69). Future studies will be required to fully address this question; however, regardless of the exact mechanism involved, our results indicate that in addition to the well-described role of donor CD8 CTL in the initial (<day 14) prevention of autoantibody production, they may also be required long-term for continued surveillance and elimination autoantibody-producing host B cells.

The mAb used in our studies is not available for human use; however, two different agonist CD40 mAb are in clinical trials as tumor therapy (70, 71). These mAb have been generally well tolerated, although their ability to promote CTL has not been fully addressed. Clearly, much further work is needed to determine whether the specific approach of CD40 stimulation will ultimately be a useful therapeutic adjunct in human lupus. Regardless of the outcome of such studies using CD40 stimulation, our results support the general concept that in vivo Ag-independent therapeutic CTL promotion is both feasible and beneficial in the setting of T cell-driven B cell hyperactivity, and support further study of this therapeutic approach in lupus.


   Acknowledgments
 
We thank Phuong Nguyen for expert technical assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants RO1 AI047466 (to C.S.V.) and RO1 CA95572 (to W.M.). Back

2 Address correspondence and reprint requests to Dr. Charles S. Via, Room B3-100, Department of Pathology, Uniformed Services University of Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814. E-mail address: cvia{at}usuhs.mil Back

3 Abbreviations used in this paper: pfp, perforin; DC, dendritic cell; FasL, Fas ligand; GN, glomerulonephritis; GVHD, graft-vs-host disease; IVCCA, in vivo cytokine capture assay; P->F1, parent-into-F1. Back

Received for publication July 10, 2007. Accepted for publication April 16, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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