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The Journal of Immunology, 2003, 170: 1667-1674.
Copyright © 2003 by The American Association of Immunologists

CD40 Ligand in Pathogenesis of Autoimmune Ovarian Disease of Day 3-Thymectomized Mice: Implication for CD40 Ligand Antibody Therapy 1

Colin Sharp2,3,*, Claire Thompson2,4,*, Eileen T. Samy*, Randolph Noelle{dagger} and Kenneth S. K. Tung5,*

* Department of Pathology, University of Virginia, Charlottesville, VA 22908; and {dagger} Department of Microbiology, Dartmouth School of Medicine, Hanover, NH 03756


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The blockade of CD40 ligand (CD40L) is effective in autoimmune disease prevention. Recently, a brief period of CD40L mAb treatment was reported to induce tolerance and enhancement of CD4+CD25+ regulatory T cell activity. We therefore determined the efficacy of CD40L mAb treatment in autoimmunity that resulted from CD4+CD25+ regulatory T cell deficiency. Autoimmune ovarian disease (AOD) and oocyte autoantibody response of day 3-thymectomized (d3tx) mice were inhibited by continuous CD40L mAb treatment from day 3, or from days 10–14, whereas CD40L mAb treatment confined to the neonatal week was ineffective. The enhanced expression of memory markers (CD44 and CD62Llow) on CD4+ T cells of the d3tx mice was unaffected by CD40L mAb treatment. In contrast, their increased T cell activation markers (CD69 and CD25) were eliminated by CD40L mAb treatment. Moreover, ex vivo activated T cells of d3tx mice expressed elevated intracellular IFN-{gamma}, and this was also blocked by CD40L mAb. The memory T cells, although nonpathogenic in CD40L mAb-positive environment, transferred severe AOD to CD40L mAb- neonatal recipients. Most importantly, CD40L mAb treatment inhibited AOD in recipients of T cells from d3tx donors with severe AOD and led to regression of AOD in d3tx mice documented at 4 wk. Therefore, 1) the continuous presence of CD40L mAb both prevents and causes regression of AOD in the d3tx mice; and 2) the multiple steps of the d3tx autoimmune disease, including T cell activation, cytokine production, T cell-mediated inflammation, and tissue injury, are CD40L dependent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The activation of T cells in response to their cognate peptide/MHC targets requires costimulatory signals delivered by APCs occurring at multiple steps (reviewed in Ref. 1). Initial naive T cell activation depends on interactions involving CD28 and B7. A second level of activation results in acquisition of T cell function and/or development of memory T cells. Finally, expression of the effector function of the activated T cells with target Ag requires costimulation that involves CD40 ligand (CD40L)6 and CD40 (reviewed in Ref. 2). Blockade of costimulation is therefore an attractive therapeutic approach for treatment of T cell-dependent autoimmune disease and allograft rejection. However, patients diagnosed with autoimmune disease already have ongoing autoimmune T cell responses; therefore, effective treatment must impact on the late stages of T cell response and silencing of preactivated or memory T cells. For this reason, effort has been devoted to the effectiveness of CD40L blockade in treatment of experimental and human autoimmune diseases.

Most studies on disease prevention have used experimental models involving immunization with protein or peptide Ags in adjuvant in which the precise timing of antigenic stimulation is known (3, 4, 5, 6, 7, 8). However, spontaneous autoimmune diseases are also responsive to CD40L mAb treatment. In the experimental lupus model of the (SWR x NZM)F1 mice, autoantibody production and glomerulonephritis were suppressed when CD40L mAb was given as a short course early in life (9). However, despite the impressive therapeutic efficacy of this treatment, the mechanism of CD40L blockade in autoimmune disease therapy is not fully understood. Moreover, the therapeutic effectiveness on subjects with ongoing autoimmune disease has not been extensively investigated.

It is now generally accepted that normal mice and rats possess regulatory T cells that are enriched within the CD4+CD25+ population. These cells have been shown to prevent the proliferation of CD25- T cells in vitro and to prevent autoimmune disease and colitis models in vivo (reviewed in Refs. 10, 11, 12). Regulatory T cell deficiency is therefore a likely pathway of spontaneous human autoimmune disease. Given this consideration, it is of interest to note that CD40L blockade was found to enhance CD4+CD25+ T cell function, and that CD40L mAb treatment may operate by induction of tolerance through activation of the regulatory T cells (13, 14). It is therefore important to investigate whether CD40L mAb treatment is effective in autoimmune diseases that are associated with CD4+CD25+ T cell deficiency. Experimentally, (C57BL/6 x A/J)F1 (B6AF1) mice thymectomized on day 3 (d3tx) after birth result in autoimmune ovarian disease (AOD), which develops at 3–4 wk. This disease can be prevented by infusion of a small number of CD4+CD25+ T cells before the d3tx mice reach 10 days of age (15). This autoimmune disease is driven by endogenous ovarian Ags (16, 17), and CD4+ T cells from d3tx mice with AOD can adoptively transfer the ovarian disease to naive recipients (16). The profound lymphopenic state of d3tx mice also contributes to autoimmune disease development (18 ; reviewed in Refs. 19 and 20). However, at present, information on the nature of the T cell response and costimulation requirements of this disease model is limited, and the response to CD40L blockade of autoimmune disease due to regulatory T cell deficiency has not yet been explored.

In this study, we investigated the effects of CD40L mAb treatment on the development and progression of AOD in d3tx B6AF1 mice. The goals of our study are 2-fold: first, to determine the preventive and curative potential of CD40L mAb treatment in an autoimmune disease associated with regulatory T cell deficiency, and second, to gain insight into the role of CD40L in the pathogenesis of AOD that follows d3tx.


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

A breeding colony of female C57BL/6 and male A/J mice purchased from National Cancer Institute (Frederick, MD) was used to generate B6AF1 progeny utilized in all following experiments. All mice were handled and kept in accordance with approved National Institutes of Health guidelines. Thymectomy was performed by the suction technique on 3-day-old female mice anesthetized by hypothermia, as described (21). Completeness of d3tx was verified histologically, and mice with residual thymus were excluded from analysis. Sham thymectomy control mice underwent the same procedure on day 3 without removal of the thymus. Ovariectomy and ovarian implantation were performed on 4-wk-old mice anesthetized with tribromoethanol. One ovary was removed, and a normal age-matched ovary was implanted under the kidney capsule through a single posterior incision.

Abs and CD40L mAb treatment

Anti-CD40L mAb (MR1) has been previously described (22). Control polyclonal hamster IgG was purchased from Pierce (Rockford, IL). For flow cytometric analysis, purified anti-CD16/CD32 (2.4G2), FITC-conjugated anti-CD62 ligand (CD62L) (MEL-14), anti-CD44 (IM7), anti-CD25 (7D4), anti-CD69 (H1.2F3), rat IgG2a (R35-95), rat IgG2b (R35-38), rat IgM (R4-22), hamster IgG (A19-3), PE-conjugated anti-CD4 (L3T4), anti-IFN-{gamma} (XMG1.2), anti-TNF-{alpha} (MP6-XT22), anti-IL-4 (11B11), anti-IL-5 (TRFK5), rat IgG1 (R3-34), rat IgG2a (R35-95), Cy-Chrome-conjugated anti-CD4 (L3T4), and rat IgG2a (R35-95) were purchased from BD PharMingen (San Diego, CA).

Anti-CD40L Ab or control hamster IgG was administered i.p. to d3tx mice every 4 days from the beginning of treatment (see Results) until the mice were sacrificed at 6 wk of age. The dose of Ab used was adjusted for the size of the mice based on their age: 25 µg/mouse, days 0–3; 50 µg/mouse, days 4–11; 100 µg/mouse, days 12–23; 150 µg/mouse, days 24–27; and 200 µg/mouse, days 28–42.

Histology and disease grading

Ovaries and residual thymus were collected in Bouin’s fixative and embedded in paraffin. Five-micrometer-thick serial sections were cut and stained with H&E. Ovarian pathology was read as unknown samples, and graded on a scale of 1–4 (21). Grade 1 inflammation consists of one to two foci of inflammatory cells, including lymphocytes, granulocytes, and monocytes restricted mainly to the hilar region. Grade 4 disease involves diffuse inflammation involving both follicles and interstitial space. Grades 2 and 3 represent intermediate inflammation. Atrophy was defined as loss of growing or mature ovarian follicles and/or loss of primordial oocytes.

Immunoperoxidase staining

Immunoperoxidase staining was used to identify the ovarian infiltrating cells. Ovaries were collected in 4% paraformaldehyde, and 5-µm-thick frozen sections were incubated with Ab to CD5 (53-7.313) for T cells, MHC class II (M5/114.15.2), or macrophages (F4/80). After washing in PBS, the sections were stained with biotinylated rabbit anti-rat Ab. Cell-bound Ab was detected by avidin-biotinylated enzyme complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA), followed by diaminobenzidine substrate (Biogenex, San Ramon, CA). The sections were subsequently counterstained with methylene blue, rinsed with water, and dehydrated with ascending concentrations of ethanol.

Oocyte autoantibody detection

Normal ovaries were collected in liquid nitrogen, and sections were cut on a cryostat and fixed in 95% ethanol. All sera were tested at a 1/50 dilution. The second Ab used was FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). Slides were viewed with a fluorescence microscope. Sera that stained the ovarian oocytes positively at the 1/50 dilution were considered positive for serum oocyte Ab.

Disease transfer

Spleens were removed from mice at 6 wk. Spleen cells were dissociated manually through nylon mesh cell strainers and briefly incubated with Tris-ammonium chloride to lyse erythrocytes. A total of 2 x 107 cells in 100 µl of sterile HBSS were injected into 7-day-old naive pups. Recipient mice were sacrificed 10 days later for study of ovarian pathology.

Flow cytometric analysis

Spleen cell suspensions (106/well) were incubated for 15 min with purified anti-CD16/CD32 Ab to block FcR binding, followed by 30 min with FITC-, PE-, and/or CyChrome-conjugated mAbs at predetermined concentrations. Isotype control Abs were used, and staining with these was subtracted as background for all data shown. Cells were acquired on FACScan and analyzed with CellQuest software (BD Biosciences, San Jose, CA). To study intracellular cytokines, spleen cell suspensions were incubated with PMA, ionomycin, and brefeldin A (Leukocyte Activation Cocktail with Golgi Plug; BD PharMingen) for 4 h, harvested, and surface stained with anti-CD4-CyC, as described above. Samples were then fixed and permeabilized with a fomaldehyde/saponin solution (Fix/Perm Buffer; BD PharMingen), washed, and stained with PE-conjugated anti-cytokine or control Abs. Cells were washed in buffer containing saponin, and the data were acquired and analyzed, as above.

Statistical analysis

Student’s t test and {chi}2 analysis were used for data analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD40L mAb administration prevents d3tx-induced AOD

To determine the effect of CD40L mAb treatment on d3tx-induced AOD, B6AF1 mice were treated continuously from the day of birth or the day of thymectomy. At 6 wk, d3tx mice treated with CD40L mAb had significantly lower incidence of AOD, with only 7 of the 35 (20%) treated mice studied showing any signs of disease as compared with 25 of 30 d3tx mice (83%) (p < 0.0001) (Table I).


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  		Table I. Effect of CD40L mAb treatment on AOD incidence and severity and oocyte autoantibody incidence in d3tx mice

 
AOD in d3tx mice can be inhibited by infusion of CD4+CD25+ regulatory cells from normal adults; however, this is effective only if cell transfer occurs before the d3tx mice reach 10 days of age (23). This finding, supported by the observation that early endogenous ovarian Ag is required for disease induction in d3tx mice (17), suggests that effector T cell response may already be triggered by day 10. We therefore determined the effect of CD40L mAb treatment initiated on day 10 or 14. Again, continuous treatment from this time was effective, with 7 of 15 (47%) CD40L mAb-treated mice getting disease (p = 0.04) (Table I).

The pathologic changes of AOD in the mice treated continuously with CD40L mAb were mild compared with those of d3tx mice (for days 0–3 group, p < 0.0001; for days 10–14 group, p < 0.0004) (Table I; Fig. 1, A and B). Ovarian atrophy, the terminal stage of AOD with loss of ovarian follicles (Fig. 1B), was detected in only 1 of 7 (14%) mice treated with CD40L mAb from days 0–3, and in none of the 14 (0%) mice treated from days 10–14, and these incidences are significantly different from those of d3tx mice (60%) (p = 0.03 and <0.03, respectively) (Table I). In contrast to CD40L mAb treatment, d3tx mice injected with hamster IgG from days 0–3 or days 10–14 had comparable incidences of AOD (81%) and ovarian atrophy as that of d3tx mice (83%) (p = 0.8–0.9). In addition, treatment with CD40L mAb led to a dramatic reduction in the extent of ovarian infiltration of T cells, MHC class II-positive cells, and F4/80-positive cells in the d3tx mice (Fig. 1, C–H).



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  		FIGURE 1. Immunopathology of AOD in d3tx B6AF1 mice. Normal ovary containing many oocytes from a mouse treated from day of birth with CD40L mAb for 6 wk. (A), Compared with the atrophic and inflamed ovary of a d3tx mouse treated with hamster IgG for the same period (B). Immunohistological detection of inflammatory cells in a d3tx mouse treated with CD40L mAb from day 10 (C, E, and G, which are sequential sections) is compared with the finding in a d3tx mouse treated with hamster IgG (D, F, and H, which are sequential sections). C and D, CD40L mAb treatment eliminates T cell infiltration (C), whereas numerous CD5+ T cells are detected in and between ovarian follicles in the d3tx mouse treated with hamster IgG (D). E and F, Treatment with CD40L mAb reduces MHC class II-positive cells (E), whereas the ovary of d3tx mouse treated with hamster IgG contains heavy infiltration of MHC class II-positive cells (F). G and H, D3tx mouse treated with CD40L mAb has the normal number of F4/80-positive macrophages distributed in the interstitium and the theca layer (G), whereas F4/80-positive cells in hamster IgG-treated d3tx mice are increased, many having entered the ovarian follicles (x200).

 
The T cell-dependent IgG Ab response critically depends on CD40L/CD40 interaction (2, 24). In this study, CD40L mAb treatment starting before disease induction significantly reduced the incidence of oocyte IgG autoantibodies among CD40L mAb-treated mice, with only 2 of 32 (6%) sera giving a positive result vs 18 of 23 (78%) sera from d3tx mice (p < 0.0001) and 16 of 24 (67%) hamster IgG-treated d3tx mice (p = 0.005). Delaying the start of treatment until day 10 or 14 yielded similar results (1 of 15 (7%) vs 12 of 16 (75%) control mice (p = 0.0006)). Also, as expected, the lymphoid tissues of mice treated with CD40L mAb were devoid of peanut agglutinin-positive germinal centers (data not shown).

Previous studies in SNF1 lupus mice and mice with experimental autoimmune encephalomyelitis (EAE) suggest that even a short course of CD40L mAb treatment early in life confers long-term suppressive effects on the disease (5, 9). It is therefore possible that CD40L mAb might have induced a state of self tolerance. To investigate whether this effect is manifested in mice with deficiency in CD4+CD25+ T cells, we treated d3tx mice with CD40L mAb from days 0–6. As shown in Table I, two injections of CD40L mAb in the neonatal week had no effect on the severity and incidence of AOD or oocyte autoantibody response when evaluated at 6 wk. Therefore, continuous CD40L mAb treatment is required to reduce AOD and autoantibody response in d3tx mice.

CD40L mAb-treated mice have reduced numbers of recently activated cells that produce IFN-{gamma}

We next determined the T cell phenotype in d3tx mice treated continuously with CD40L mAb. Similar to d3tx mice, the majority of CD4+ cells in the d3tx mice treated with CD40L mAb for 5–6 wk displayed a CD62Llow (Fig. 2, A and C) and CD44high (Fig. 2, B and D) memory phenotype, a finding consistent with T cells having undergone homeostatic expansion in T lymphopenic mice (25, 26, 27). In contrast, the early T cell activation marker CD69 on CD4+ cells from CD40L mAb-treated mice was significantly reduced as compared with d3tx mice that were untreated or given hamster IgG (Fig. 2E) (p = 0.0007 and 0.0002, respectively). The percentage of CD4+ T cells with increased CD25 expression was significantly greater in mice with d3tx (n = 10) when compared with sham thymectomized mice (p = 7 and 0.001, respectively). Although the level of CD25+ T cells in d3tx mice treated with CD40L mAb (n = 16) appeared lower than those of d3tx mice, the change was not statistically significant (p = 0.14) (data not shown).



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  		FIGURE 2. Flow cytometric analysis of spleen CD4+ T cells from d3tx mice treated with CD40L mAb or hamster IgG, and from normal and d3tx mice. A and B, Flow cytometry profiles of a representative normal mouse (black), a d3tx mouse (blue), and a d3tx mouse treated from day 3 with CD40L mAb (red) for CD62Llow (A) and CD44high cells (B) gated on CD4+ cells. C and D, The percentage of CD62Llow (C) and the percentage of CD44high (D) CD4+ T cells from d3tx mice treated with CD40L mAb (red) do not differ from those of d3tx mice (blue) or d3tx mice treated with hamster IgG ({circ}). E and F, The percentages of CD4+ T cells with CD69 (E) and intracellular IFN-{gamma} (F) are significantly higher in d3tx mice compared with normal mice, whereas CD40L mAb, but not hamster IgG treatment effectively reduces the percentage of IFN-{gamma}-positive cells to normal. G and H, Cytometric analysis of representative individual mice, showing higher numbers of IFN-{gamma}-positive CD4+ cells in a d3tx mouse treated with hamster IgG (G) compared with the finding in a mouse treated with CD40L mAb (H).

 
To determine change in T cell function, we analyzed the splenic CD4+ T cells for intracellular cytokine production after ex vivo activation. Compared with normal mice, d3tx mice had significantly higher number of CD4+ cells that stained positively for intracellular IFN-{gamma} (Fig. 2, F–H). Following CD40L mAb, but not hamster IgG treatment, the percentage of IFN-{gamma}-producing CD4+ T cells was completely reversed to that of normal mice (p = 0.002) (Fig. 2F). In contrast, similar percentages of intracellular TNF-{alpha}-positive CD4+ T cells were detected in normal mice, d3tx mice, and d3tx mice treated with CD40L mAb; and IL-4 and IL-5 were not detected in the CD4+ cells from any of the groups tested (data not shown).

Spleens from CD40L mAb-treated d3tx mice contain pathogenic cells and retain the ability to transfer AOD

In this study, we determined the in vivo function of T cells from d3tx mice treated with CD40L mAb by their capacity to transfer AOD to normal B6AF1 recipients. This also addresses the question of whether the pathogenic T cells in CD40L mAb-treated d3tx mice have been deleted or rendered anergic. A total of 2 x 107 splenocytes from 6-wk-old d3tx mice treated from birth or from day 3 were transferred into normal B6AF1 recipients less than 7 days of age. Surprisingly, although the cell donors had little or no ovarian disease themselves, their spleen cells transferred severe AOD. The disease incidence in the cell recipients was similar to those found in recipients of T cells from d3tx donors (p = 0.8) or from d3tx donors injected with hamster IgG (p = 0.9) (Fig. 3). Therefore, CD40L mAb treatment did not delete or irreversibly incapacitate the pathogenic T cell function.



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  		FIGURE 3. AOD of comparable severity is found in recipients of spleen cells from syngeneic d3tx donors that are untreated and treated for 5–6 wk with CD40L mAb or hamster IgG. A total of 2 x 107 cells are transferred into recipients less than 7 days old, and the ovarian pathology determined 10 days later.

 
Although our findings suggest that CD40L mAb might inhibit AOD by blocking CD4+ T cell activation, as shown by reduction in CD69 expression and IFN-{gamma} production, CD40L mAb treatment may also act on later stages of AOD induction in d3tx mice. This possibility is examined in the next study.

Activated pathogenic T cells can be prevented from causing disease by CD40L mAb administration

A total of 2 x 107 splenocytes from 6-wk-old, d3tx mice were transferred into young, naive, syngeneic recipients. The recipients were then given either CD40L mAb or control hamster IgG on days 0, 4, and 8 after cell transfer and were assessed for disease on day 10. As shown in Fig. 4, treatment with CD40L mAb, but not control hamster IgG, significantly reduced the incidence and severity of disease of the spleen cell recipients (p = 0.008). Therefore, CD40L mAb prevents activated T cells from expressing their pathogenic potential. The finding that CD40L blockade inhibits activated or memory T cell response prompted the next study, in which we examined the therapeutic potential of CD40L mAb in mice with histologically documented AOD.



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  		FIGURE 4. The severity of AOD in spleen cell recipients from 6-wk-old d3tx donors is reduced by treatment of recipients with CD40L mAb, but not with hamster IgG.

 
Established AOD in d3tx mice is reversed by CD40L mAb treatment

To determine the response of ongoing AOD to CD40L mAb treatment, one ovary from each d3tx mouse was studied at 4 wk, when most mice have developed AOD (17). Following unilateral ovariectomy, each mouse was implanted with an age-matched normal ovary to maintain the endogenous Ag load. The mice were then treated with CD40L mAb or control hamster IgG every 4 days for the next 2 wk. At 6 wk, the pathology of the second endogenous ovary as well as that of the ovarian graft was determined.

CD40L mAb treatment from 4 to 6 wk resulted in the resolution or reduction of ovarian inflammation in 11 of the 14 mice (p = 0.006) (Fig. 5, A, D, and E). Of the three mice that did not change in pathology grades, one had terminal ovarian atrophy by 4 wk, and another was unresponsive to d3tx. In contrast, 7 of the 8 hamster IgG-treated d3tx mice had more severe inflammation in the ovary taken at 6 wk than that taken at 4 (p = 0.02) (Fig. 5, B, F, and G). The mouse without disease progression did not have AOD at any time. Study of the ovarian grafts showed a similar degree of inflammation as the endogenous ovary taken at the same time (data not shown).



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  		FIGURE 5. CD40L mAb treatment from 4 to 6 wk reduces the severity of ongoing AOD and oocyte autoantibody response in d3tx mice. In this study, the pathology in two ovaries from the same mice from 4 to 6 wk was compared. A, In 11 of 14 mice treated with CD40L mAb, the AOD scores decline from 4 to 6 wk. B, The AOD scores of essentially all the mice treated with hamster IgG increase from 4 to 6 wk. C, The CD40L mAb treatment reduces the incidence and the titer of oocyte autoantibodies detectable at 6 wk. These mice did not have detectable serum oocyte autoantibody at 4 wk. D and E, Pathology in the two ovaries from a d3tx mouse treated with CD40L mAb. The ovary at 4 wk contained multiple foci of mononuclear inflammatory cellular infiltrates (arrows) (D). After treatment with CD40L mAb for 2 wk, the second ovary was free of inflammation (E). F and G, Pathology in two ovaries of a d3tx mouse treated with hamster IgG. The ovary at 4 wk contained two loci of inflammation (arrows) (F). After hamster IgG treatment, the second ovary progressed to complete atrophy with heavy monocytic inflammation (G). H&E, x200.

 
In parallel with AOD reduction, the oocyte autoantibody response of the d3tx mice treated with CD40L mAb was also significantly reduced. Serum oocyte autoantibody was not detected in any of the mice studied at 4 wk, but by 6 wk, the CD40L mAb-treated mice showed a reduction in oocyte autoantibody titer compared with control d3tx mice injected with hamster IgG (p = 0.01) (Fig. 5C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrated that CD40L blockade by CD40 mAb administration prevents the development of AOD and oocyte autoantibody response in d3tx B6AF1 mice. The treatment was effective regardless of whether CD40L mAb was started before day 3, or at days 10–14, when T cell activation by endogenous Ag has most likely manifested (17). However, disease inhibition requires the continuous treatment with CD40L mAb, and treatment with CD40L mAb confined to the neonatal week was not effective. Most importantly, the study has clearly documented a potent therapeutic effect of CD40L mAb for d3tx-induced autoimmune disease. Essentially, all d3tx mice with histologically documented AOD responded to a 2-wk course of CD40L mAb treatment, leading to reduction or complete regression of the ovarian pathology. Concomitantly, there was reduction (or less likely, prevention) of their autoantibody response. In addition, the study has identified the steps in AOD development that are susceptible to CD40L blockade and provided new insights on the mechanism of therapeutic action of CD40L mAb.

The CD40L/CD40 interaction has been shown to operate at many critical steps in the immune response and the inflammatory response. In autoimmune disease context, CD40L blockade may inhibit disease by induction of tolerance when the mAb is given during disease induction, or by inhibition of an ongoing autoimmune response. Recent studies suggested that CD40L engagement may induce tolerance to alloantigen by expansion and/or activation of Ag-specific CD4+CD25+ regulatory T cells (13, 14). This may explain why a short course of CD40L mAb treatment was sufficient to control autoimmune diseases such as EAE and murine lupus in the (SWR x NZM)F1 mice. It may also explain why continuous CD40L mAb treatment is required for prevention of AOD in the d3tx mice, because CD4+CD25+ T cells are depleted in these mice. In this regard, it has been shown that colitis, induced by transfer of CD45RBhigh CD4+ T cells into SCID mice deficient in regulatory T cells, also requires continuous CD40L mAb treatment (28). Therefore, in treatment of human autoimmune disease, the strategy of CD40L mAb administration should take into consideration whether the underlying disease mechanism involves a deficiency in regulatory T cells.

D3tx results in profound T lymphopenia. The up-regulation of CD44 expression and reduction of CD62L expression on the CD4+ T cells from the d3tx mice support the possibility that the lymphopenic state is associated with compensatory proliferation of the CD4+ T cells (29, 30, 31). In d3tx mice treated with CD40L mAb, the expression of CD44 and CD62L was unaltered. Therefore, CD40L did not affect AOD development through prevention of compensatory T cell proliferation attendant to d3tx. This is consistent with previous reports that compensatory T cell proliferation occurs normally in mice deficient in CD40 (32). D3tx mice also exhibit increased expression of CD69 and CD25 on CD4+ T cells, providing evidence for polyclonal T cell activation (29). In this study, CD40L mAb treatment was found to reduce the CD69 on CD4+ T cells to the level of normal mice. More importantly, this study has now documented that ex vivo activated CD4+ T cells of d3tx mice had significant increase in intracellular IFN-{gamma} production, and this change was completely reversed by CD40L mAb treatment. Although T cell activation is reduced by CD40L mAb treatment, it is unclear whether this alone is sufficient to prevent AOD development. This is because the T cell activation event may or may not represent, or include, the specific T cell response to endogenous ovarian Ag required for AOD induction. In addition, as will be described below, CD40L mAb blocks additional downstream event(s) in AOD induction, and the multiple effects of CD40L blockade are not easily dissociable. Nevertheless, because CD40L is primarily required for Th1 rather than Th2 response (33), the finding that AOD and CD4+ T cell production of IFN-{gamma} in the d3tx mice are both inhibited by CD40L blockade suggests that the Th1 response is most likely an important effector mechanism in AOD of the d3tx mice.

The inhibition of T cell activation in the d3tx host appears to be reversible and is dependent on continuous presence of CD40L mAb. When the T cells from CD40L mAb-treated d3tx mice were directly transferred to the CD40L mAb-negative environment of normal syngeneic recipients, they acquired pathogenic potential and induced severe AOD. Although the ability of T cells of CD40L mAb-treated mice to transfer disease has been reported in previous studies on AOD induced by a zona pellucida 3 peptide and to lesser extent in EAE (4, 5), both peptide/adjuvant models require ex vivo T cell activation before adoptive transfer. It is therefore unclear whether the T cells acquire pathogenic capacity during in vitro stimulation, or in vivo, within the recipient. In the case of AOD, the T cells from CD40L mAb-treated d3tx mice, activated ex vivo, produced IFN-{gamma} in comparable levels to normal mice. However, when transferred in vivo, they were able to traffic to the target organ and cause disease. This suggests that potentially pathogenic T cells persist in the CD40L mAb-treated mice, which can then become activated when CD40/CD40L interaction is allowed to proceed.

Treatment of cell recipients with CD40L mAb effectively inhibited the adoptive transfer of AOD by activated or memory T cells from d3tx donors. This finding provides the basis for investigation on the therapeutic property of CD40L mAb. Inhibition of disease transfer of CD40L mAb delivered to cell recipients was also observed in mice with experimental autoimmune thyroiditis (6) and in EAE (5). The effect of CD40L mAb on the late event(s) of AOD induction could include T cell trafficking to the target organ, interaction with the cognate Ag, response to the APC in target organ, and/or further activation and production of proinflammatory cytokines by the activated T cells. In a recent study on EAE, the analysis of donor cells bearing the Thy-1.1 marker in the spinal cord recorded similar numbers between Ab-treated and control mice early in disease transfer, but the numbers were significantly higher in the control mice 2 days later (34). This finding suggests that CD40L mAb inhibits effector T cell proliferation in, or their recruitment to, the target organ.

In the study on AOD, we can directly investigate the therapeutic efficacy of CD40L mAb treatment by comparing the presence and extent of histopathology in the paired organs from the same animal at two time points. Because endogenous ovarian Ag is required to maintain the progression of AOD in d3tx mice (17), we implanted an ovarian graft to maintain a constant endogenous ovarian Ag load. Using this approach, our study clearly documents the therapeutic efficacy of CD40L mAb on >90% of the mice that have developed AOD. Previous studies also suggested a therapeutic potential of CD40L mAb. In EAE, CD40L mAb treatment in mice with clinical paralysis was found to block disease progression (5). However, EAE is a disease associated with relapses and remissions, and disease relapse can result from epitope spreading that depends on de novo T cell response to new antigenic epitopes of the myelin basic protein or other encephalitogenic proteins (35, 36). Thus, the response of mice with EAE to CD40L mAb might be due to inhibition of disease relapse and hence de novo EAE induction, rather than regression of ongoing EAE. In murine lupus, the precise timing of Ag stimulation driving the complex immunopathologic processes of systemic lupus is unclear; therefore, it is not possible to determine whether the inhibitory effect on autoantibody production was conferred before or after pathogenic T cell response (9). In contrast, treatment of nonobese diabetic mice with insulitis did not demonstrate beneficial effect on development of diabetes mellitus (37). It is possible that CD40L mAb would be effective if administered before the target antigenic cells are destroyed. Thus, the d3tx mouse in Fig. 5A that had atrophic ovary before treatment did not respond to CD40L mAb, and this may also explain why nonobese diabetic mice with insulitis did not respond to CD40L mAb treatment (37).

In summary, continuous CD40L mAb treatment has been documented to prevent AOD development and to induce the regression of established AOD in d3tx mice. The functional requirement of CD40L costimulation in d3tx-induced autoimmune disease appears to occur at multiple steps, including T cell activation and the functional expression of the activated T cells. In addition, the therapeutic efficacy of the CD40L mAb treatment most likely depends on blockade of CD40L requirement late in disease induction, beyond T cell activation. In contrast, unlike other autoimmune models, a short course of CD40L mAb treatment was not effective. These findings should impact on the design of regimen in clinical autoimmune disease or allograft rejection based on CD40L mAb treatment.


   Acknowledgments
 
We thank Sharon Mangawang for expert assistance in neonatal thymectomy and histology.


   Footnotes
 
1 The study is supported by National Institutes of Health Grants R01 AI-41236, AI-51420, and P50 AR-45222. Back

2 C.S. and C.T. contributed equally to this work. Back

3 Current address: Laboratory for Clinical and Molecular Virology, Royal (Dick), School of Veterinary Studies, University of Edinburgh, Edinburgh, U.K., EH9 1QH. Back

4 Current address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, U.K., OX1 3RE. Back

5 Address correspondence and reprint requests to Dr. Kenneth S. K. Tung, Department of Pathology, P.O. Box 800214, University of Virginia, Charlottesville, VA 22908. E-mail address: kst7k{at}virginia.edu Back

6 Abbreviations used in this paper: CD40L, CD40 ligand; AOD, autoimmune ovarian disease; CD62L, CD62 ligand; d3tx, day 3 thymectomized; EAE, experimental autoimmune encephalomyelitis. Back

Received for publication October 9, 2002. Accepted for publication November 13, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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