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Ligation of CD28 In Vivo Induces CD40 Ligand Expression and Promotes B Cell Survival¹

Deling Yin^*, Liying Zhang^*, Ruoxiang Wang^*, Laszlo Radvanyi^2,§, Christian Haudenschild, Qiding Fang^*, Marilyn R. Kehry and Yufang Shi^3,*

Departments of ^* Immunology and Experimental Pathology, Jerome H. Holland Laboratory, American Red Cross, Rockville, MD 20855; Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877; and ^§ Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Canada

	Abstract

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Functional activation of T cells requires ligation of Ag receptors with specific peptides presented by MHC molecules on APCs concurrent with appropriate contacts of cell surface accessory molecules. Among these accessory molecules, interactions between CD28/CTLA-4 with B7 family members (CD80 and CD86) and CD40 with CD40 ligand (CD40L) play a decisive role in regulating the progression of balanced immune responses. However, most information regarding the role of accessory molecules in immune responses has been derived in the context of signals from the TCRs. Little understanding has been achieved regarding the consequence of ligation of costimulation molecules in absence of signals from the TCR. By employing an in vivo murine system, we show, herein, that ligation of CD28 alone with anti-CD28 Abs leads to a dramatic enlargement of the peripheral lymphoid organs characterized primarily by the expansion of B cells. B cells from anti-CD28-treated mice are resistant to spontaneous and anti-IgM-induced apoptosis. These cells are also unsusceptible to FasL-mediated apoptosis. Interestingly, this in vivo effect of CD28 on B cells is largely mediated by inducing the expression of CD40L, since coadministration of a blocking Ab against CD40L inhibited CD28-mediated B cell survival and expansion. Therefore, CD28-mediated expression of CD40L may play an important role in the regulation of lymphocyte homeostasis.

	Introduction

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In the absence of proper costimulation, activation of T cells through their Ag receptors not only leads to their inability to proliferate and to produce cytokines, but also renders T cells unresponsive to further activation, a phenomenon referred to as anergy (1, 2). Among many T cell costimulatory molecules identified to date, CD28 is by far the most potent one (3, 4). CD28 is a member of the Ig gene superfamily and is expressed on the T cell surface as a disulfide-linked homodimer of 44-kDa glycoprotein subunits (5, 6). CD28 has been shown to synergize with the mitogenic signals from the TCR by interacting with two related cell surface molecules, CD80 (BB1; B7.1) and CD86 (B7.2) on APCs (7, 8). In addition, CTLA-4, another accessory molecule on the T cell surface, also binds to CD80 and CD86 with a much higher affinity than CD28 (9). Ligation of CTLA-4, in contrast to CD28, has been shown to exert a negative signal on T cell activation (10). It has been proposed that CD28 and CTLA-4 antagonize each other and that their differential expression patterns provide an important mechanism for balancing immune responses (10). Blockade of CD80 and CD86 with CTLA-4-Ig fusion protein, which inhibits both CD28 and CTLA-4, has been shown to prolong the survival of xenografts and allografts in animal models (11). Though the disruption of the CD28 gene does not affect the development and the selection of T cells in mice, mature T cells from these mice have impaired responses to lectins (12). Furthermore, CD28-deficient mice exhibit defects in Ab isotype switching and are more susceptible to pathogens that depend on an intact humoral response (12). CD28-deficient mice also exhibit decreased B cell activation and Ab production to various Ags (13). The germinal center B cells in CD28-deficient mice do not undergo proliferative expansion in response to antigenic challenge (14). Therefore, though CD28 is expressed on T cells, this molecule also profoundly affects B cells.

Except secreting cytokines, T cells also influence B cells via membrane-associated molecules (15). One of the best-characterized pathways by which T and B cells interact is by direct interaction between CD40 and CD40 ligand (CD40L)⁴ (16, 17). CD40 is constitutively expressed on B cells, and CD40L is transiently expressed on activated T cells. It has been shown that ligation of CD40 could promote Ig class switch (18), B cell proliferation, expression of Fas (19), and cell viability (20). Thus, the level of CD40L expressed on T cells is an important mechanism by which T cells regulate B cell responses. It has been shown that activation of T cells via the TCR could induce the expression of CD40L (21). It is also known that some cytokines could induce the expression of CD40L. However, the exact effects of the ligation of accessory molecules alone on the expression of CD40L have yet to be elucidated.

Though it has been suggested that CD40L expression is solely dependent on TCR signals (22), several studies have demonstrated that costimulation through CD28 enhances T cell-dependent B cell activation via CD40-CD40L interaction when TCR is engaged (23, 24, 25, 26). As a consequence, T cell-dependent B cell growth and differentiation were consistently augmented, when anti-CD3-stimulated T cells were simultaneously activated with anti-CD28 (27). This T cell-mediated B cell growth was found to be dependent on CD28 costimulation-induced increases of CD40L on T cells. However, it is not known whether the increase in CD40L expression on T cells is due to CD28 ligation alone, or also requires TCR ligation. We have studied the effect of ligation of CD28 in vivo by administration of anti-CD28 Abs. We have previously shown that this treatment could inhibit activation-induced apoptosis in T cells (28). We report herein that administration of anti-CD28 induces the expression of CD40L on T cells and splenomegaly characterized by the expansion of B cells. These B cells are resistant to spontaneous and activation-induced apoptosis. The expansion of B cells could be inhibited by coinjection of a blocking Ab to CD40L. Therefore, CD28 ligation in absence of exogenous Ags induces the expression of CD40L, which in turn regulates B cell homeostasis.

	Materials and Methods

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Mice and Abs

Four- to 6-wk-old male BALB/c mice were obtained from the National Cancer Institute (Frederick, MD) and were maintained in the vivarium of the Holland Laboratory of the American Red Cross, a facility accredited by the American Association for the Accreditation of Laboratory Animal Care, Inc. (AAALAC). CD28 knockout mice were kindly provided by Dr. Tak Mak (Ontario Cancer Institute, Toronto, Canada). Mice were allowed to acclimatize to the new environment for at least one week after shipping. Animals were age matched in each experiment.

Anti-CD28 was purchased from PharMingen (La Jolla, CA), or was produced by a B cell hybridoma, 37.51, kindly provided by Dr. James P. Allison (University of California at Berkeley, CA). Hamster anti-murine CD40L B cell hybridoma, MR1, was provided by Dr. Randolph Noelle (Dartmouth Medical School, Hanover, NH). mAbs were purified by chromatography with protein G-coupled sephadex. In some cases, hybridoma culture supernatant was precipitated with saturated (NH₄)₂SO₄ (pH 7.2), redissolved in PBS, and dialyzed extensively against PBS. The anti-CD28 Ab prepared in our laboratory was assessed for the presence of endotoxin by AlerCHEK (Portland, ME) and found to have negligible amount (<2.0 EU/ml). Normal hamster Ig obtained from Sigma (St. Louis, MO) was used as a control. PE-anti-CD40L and FITC-anti-CD3 were purchased from PharMingen. FITC-labeled goat anti-murine IgM was a gift of Dr. David Scott (The Holland Laboratory of the American Red Cross, Rockville, MD).

In vivo Ab administration

Mice were injected i.p. with purified anti-CD28, normal hamster Ig, or anti-CD40L (MR1) at indicated doses and dosing regimes. Splenocytes were isolated for the assessment of apoptosis and histological studies at indicated times. Splenocytes were also analyzed for the expression of cell surface proteins by flow cytometry or Western blotting.

In vitro activation of splenocytes

Freshly isolated spleens were made into single cell suspensions by pressing them between frosted ends of two microscope slides. Splenocytes (3 x 10⁵/well) were activated with anti-IgM at 30 µg/ml (29) in 96-well tissue culture plates in 100 µl RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 2 mM glutamine, 50 µM mercaptoethanol, 50 µg/ml gentamicin, and 10% heat-inactivated FCS (Sigma). After 24 h of incubation at 37°C in the presence of 5% CO₂, cells were harvested for the assessment of genomic DNA integrity.

Fas ligand-mediated apoptosis

The sensitivity to Fas ligation-induced apoptosis in splenocytes was determined by coculturing with L cells expressing sense Fas ligand (FasL) or antisense FasL (kindly provided by Dr. T. A. Ferguson, Washington University School of Medicine, St. Louis, MO) (30). Cells were harvested at 12 h, and apoptosis was determined by flow cytometric DNA content analysis as described below.

Flow cytometry

Single splenocyte suspensions were prepared after different treatments and washed with PBS supplemented with 1% FCS and 0.02% sodium azide (staining buffer). Cells (1 x 10⁶) were stained with FITC-anti-CD3, FITC-anti-IgM, or PE-anti-CD40L (PharMingen) in staining buffer at 4°C for 30 min, washed twice with PBS, and fixed with 1% formalin in PBS. Cells were analyzed for fluorescence intensity on a FACScan flow cytometer with a single argon laser and logarithmic intensity scales using the CellQuest program (Becton Dickinson, San Jose, CA).

Flow cytometric analysis was also employed to assay cellular DNA content, where apoptotic cells were shown as a hypodiploid peak. After treatments, splenocytes were fixed with 70% ethanol for 30 min at 4°C, followed by two washes with PBS. The fixed splenocytes were then incubated in PBS containing propidium iodide (Sigma) at 50 µg/ml and RNase (Boehringer Mannheim, Indianapolis, IN) at 0.1 mg/ml at room temperature for 30 min. DNA content was determined by flow cytometry on FACScan (Becton Dickinson). The FL2 intensity was plotted as histograms on a linear scale.

Western blotting

Equal numbers of cells were lysed in RIPA lysis buffer, which was composed of 1% Nonidet P-40, 50 mM HEPES (pH 7.4), 150 mM NaCl, 500 µM orthovanadate (Fisher Scientific, Fairlawn, NJ), 50 mM ZnCl₂, 2 mM EDTA, 2 mM PMSF, 0.1% SDS, and 0.1% deoxycholate. Samples were incubated at 4°C for 10 min and then centrifuged at 10,000 x g for 15 min at 4°C. The supernatants were transferred, mixed, and boiled in SDS sample buffer. The lysates were separated by polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was then incubated at room temperature in a blocking solution composed of 5% skim milk powder dissolved in 1x TBS (10 mM Tris, pH 8.0, and 140 mM NaCl) for 1 h. The membrane was then incubated with the blocking solution containing anti-CD40L (1 µg/ml) for 4 h at room temperature. After washing three times with TBS for 5 min, the blot was then incubated with a HRP-conjugated protein A in the blocking solution. The blot was again washed three times with TBS before being exposed to ECL (Amersham, Arlington Heights, IL).

Histology sections

Spleen samples were fixed in 10% formalin in PBS and embedded in paraffin. Three-micron sections were stained with hematoxylin and eosin and viewed under light microscope, and representative areas were microphotographed.

ELISA

Total and hamster Ig-specific mouse Ab were detected by ELISA. To determine total amounts of mouse Ig, serum samples were diluted in a binding buffer (0.05 M Tris, pH 9.0) and incubated in 96-well ELISA plates. After overnight incubation at 4°C, the plates were washed with PBS plus 0.05% Tween 20, and the nonspecific binding sites were blocked with 0.5% gelatin in PBS. The amount of mouse Ig was detected by HRP conjugated to rabbit anti-mouse L chain (PharMingen). To assess the amount of Ab in mouse serum specific to hamster Ig, ELISA plates were first coated with hamster anti-mouse CD28 mAb (37.51) at 10 µg/ml in the binding buffer. After blocking of the nonspecific binding sites, the plates were further incubated with mouse serum or mouse anti-hamster Ab (Sigma) as standard. Similarly, the amount of mouse Ig binding hamster Ig was detected by HRP conjugated to rabbit anti-mouse L chain. After development with HRP substrate ABTS (Sigma), the amount of mouse Ig was determined by absorbance reading.

Cell proliferation assay

Freshly isolated splenocytes (5 x 10⁵/well) were incubated in 96-well plates with 200 µl RPMI 1640 (Life Technologies) supplemented with 10% FBS, 2 mM glutamine, 50 nM 2-ME, and 10 mM gentamicin (Life Technologies). [³H]Thymidine was added to the culture at 1 µCi/well. Cells were harvested onto glass-fiber filter paper at 6 h after culture. Cell proliferation in terms of [³H]thymidine uptake was measured by liquid scintillation counting.

	Results

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In vivo administration of anti-CD28 induces splenomegaly in mice

Previous studies have demonstrated that ligation of CD28 could inhibit activation-induced apoptosis in T cells (10, 28, 31, 32). Thus, it is conceivable that CD28 mediates cell survival signals. Indeed, others and we have shown that CD28 induces the expression of Bcl-X, a protein that increases the resistance of cells to apoptosis (10, 31). To assess whether sustained cell survival induced by treatment with anti-CD28 would alter cellular homeostasis in the peripheral lymphoid organs, we injected mice i.p. with three doses of anti-CD28 at 48-h intervals. Mice did not show any clinical abnormality over the course of the treatment. However, this treatment resulted in a marked increase in the size of the spleen and lymph nodes. The size of the spleen increased 3- to 5-fold (28), and the number of splenocytes per spleen also dramatically increased (Table I). When splenocytes of anti-CD28-treated mice were stained with anti-CD3 for T cells and anti-IgM for B cells, flow cytometric analysis revealed that there was a dramatic increase in the percentage of B cells, with a decrease in the percentage of T cells. When the total cell number in each population was calculated, the number of splenic T cells in anti-CD28 treated mice did not change significantly, while the number of splenic B cells increased 4.4-fold. Thus, the enlargement of the spleen is mainly caused by an increase in the number of B cells, suggesting that the ligation of CD28 alone in vivo modulates B cell survival.

View larger version (78K): [in this window] [in a new window]   FIGURE 1. Histological analysis of the alterations of the spleen induced by in vivo administration of anti-CD28 and the inhibitory effect of blocking CD40L. Six-wk-old mice were injected i.p. with 200 µg anti-CD28 with or without 200 µg anti-CD40L (MR1) at 48-h intervals. Mice were euthanized at 24 h after the third injection. Spleens were fixed with 10% formalin, and 3-micron sections were stained with hematoxylin and eosin. A, Normal hamster Ig. B, Anti-CD28. C, Anti-CD28 and anti-CD40L.

View larger version (48K): [in this window] [in a new window]   FIGURE 2. Anti-CD28 does not induce spleen enlargement in CD28-deficient mice. Five-wk-old CD28-deficient (CD28-/-) mice or CD28+/+ littermates were injected 3 times with 200 µg anti-CD28 at 48-h intervals. Mice were euthanized on day 7. Spleen were harvested and photographed.

Anti-CD28 in vivo induces the expression of CD40L

It has been shown that CD28 ligation enhances TCR-induced expression of CD40L on the T cell surface in vitro (23.). In addition, constitutive expression of CD80 on L cells could enhance the expression of CD40L on CD4⁺ T cells; however, the exact role of CD28 in such a system was not clear (33). Nevertheless, our system could provide further information about the role of CD28 in the regulation of CD40L in vivo. We found that, at 24 h after a single injection of anti-CD28, about 25% of the T cells (gated based on the expression of CD3) became positive for CD40L as detected by flow cytometry, while less than 1% of the T cells were positive for CD40L in control mice (Fig. 3). In addition, we also examined the expression of CD40L by Western blot analysis. After treatment with anti-CD28 or normal hamster Ig for 24 h, splenocytes were lysed, and the expression of CD40L was detected with anti-CD40L on Western blots. We found that anti-CD28 treatment increased the expression of CD40L detected as a band with the molecular mass of 32 kDa (Fig. 4). Therefore, in vivo ligation of CD28 alone could induce the expression of CD40L.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. CD28 ligation induces surface expression of CD40L. Mice were injected with 200 µg anti-CD28. The expression of CD40L on splenocytes was analyzed at 24 h with FITC-anti-CD40L by flow cytometry.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Western blot analysis of CD40L expression induced by in vivo administration of anti-CD28. Mice were treated with 200 µg anti-CD28 or 200 µg anti-CD40L for 24 h. Control mice received the same amount of normal hamster Ig. Splenocytes were isolated and lysed for Western blot. MR1 at 1 µg/ml was used as the primary Ab.

It has been shown that ligation of CD40 alone provides potent mitogenic signals to B cells. We, therefore, investigated the role of CD40L expression in the induction of B cell expansion induced by in vivo CD28 ligation. We injected mice with a blocking Ab to CD40L together with anti-CD28. We found that coadministration of anti-CD40L dramatically reduced anti-CD28-induced enlargement of peripheral lymphoid organs, while coinjection of normal hamster Ig did not have such an effect (data not shown). The effect of anti-CD40L was also observed when splenocyte number was analyzed (Table I). On histology, we found that anti-CD40L also blocked the splenic expansion of the white pulp induced by anti-CD28 (Fig. 1). Therefore, in vivo CD28 ligation-induced CD40L expression appears to be largely responsible for the alteration of lymphocyte homeostasis.

In vivo administration of anti-CD28 enhances the resistance of B cells to apoptosis

Others and we have previously established that ligation of CD28 could inhibit activation-induced apoptosis in T cells (10, 28, 31). It has been shown that CD40 ligation prevents the induction of apoptosis in B cells (20). Based on the observation that CD28 ligation could induce the expression of CD40L, we hypothesized that the effect of CD28 on B cells in vivo could be exerted by affecting the susceptibility of B cells to the induction of apoptosis, which might account for the splenic enlargement. Mice were injected i.p. with three doses of anti-CD28 at 48-h intervals. The splenocytes were harvested and treated with a high dose of anti-IgM (30 µg/ml), which has been shown to effectively induce apoptosis in primary B cells (29). Shown in Fig. 5, the splenocytes from anti-CD28-treated mice showed resistance to anti-IgM-induced apoptosis. In addition, under these culture conditions, about 20% of normal splenocytes undergo spontaneous apoptosis 12 h after culture, while no apoptosis was observed in anti-CD28 treated splenocytes.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Anti-CD28-stimulated splenocytes are resistant to high dose anti-IgM-induced B cell apoptosis. Splenocytes were isolated from mice treated with three doses of 200 µg anti-CD28 or normal hamster Ig at 48-h intervals and treated with or without 30 µg goat anti-mouse IgM for 12 h. DNA content was analyzed by flow cytometry upon staining with propidium iodide. Apoptotic cells are indicated by the degree of the hypo-diploid peak.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Anti-CD28-stimulated splenocytes are resistant to FasL-induced apoptosis. Splenocytes from mice treated with three doses of 200 µg anti-CD28, anti-CD40L, or normal hamster Ig at 48-h intervals were cocultured with L cells transfected with FasL cDNA in sense (S) or anti-sense (AS) orientations. Nonadherent cells were harvested at 12 h. DNA content was analyzed by flow cytometry after staining with propidium iodide.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results DISCUSSION References

 
Activation of costimulatory molecules is crucial for T cell activation and B cell proliferation and differentiation (34). CD40:CD40L and CD28/CTLA-4:CD80/CD86 are the best established costimulation molecule systems. T cells provide help for B cell proliferation and Ab production by cell-cell contact and by releasing soluble factors. CD40L, which constitutes contact-dependent T cell help, is the predominant B cell costimulation protein expressed on activated T cells upon activation (35). On the other hand, B cells also express molecules such as CD80 and CD86 to provide costimulation to T cells via CD28 or CTLA-4 (36). Blocking CD40-CD40L interactions with specific Ab leads to severely impaired Ab production (37). CD40L-deficient mice exhibit defects in T cell-dependent B cell responses, which could be fully reconstituted by activating Abs to CD40 (38, 39). CD40-CD40L interaction induces the expression of CD86 on B cells. The fact that cross-linking CD40 on B cells promotes expression of the ligand (CD80) for CD28 suggests that T and B interactions may have a reciprocal amplification mechanism (40). We have demonstrated here that administration of anti-CD28 mAb in vivo leads to dramatic enlargement of peripheral lymphoid organs, due primarily to an increase in the number of B cells. These B cells are resistant to spontaneous and high dose anti-IgM-induced apoptosis. Furthermore, our data show that anti-CD28 induces the expression of CD40L on T cells. Anti-CD28-induced B cell expansion is blocked by inhibitory Ab to CD40L. Thus, CD28 ligation alone can induce the expression of CD40L, which is responsible for the expansion of B cells.

CD40L is present on activated, but not resting, T cells. The expression of CD40L on T cells is tightly regulated (41, 42, 43). The modulation of CD40L expression has been demonstrated in various systems. It has been shown that CD40L expression is induced, peaking at 5 h, after T cell activation (44). It has been shown that the induction of CD40L expression in vitro and in vivo on CD4⁺ T cells is dependent on the expression of CD80. When expressed on L cells, CD80 was found to be both necessary and sufficient for the induction of CD40L on normal CD4⁺ T cells (32). This is in agreement with the data presented herein. However, when normal accessory cell populations were used, only partial inhibition of induction of the CD40L was observed with reagents that inhibit B7/CD28 interactions (32). Furthermore, CD40L could be induced on CD4⁺ T cells from CD28-deficient mice (45). Therefore, non-B7/CD28 cellular interactions can also mediate the costimulatory signals needed for induction of CD40L expression. This may explain why in our experiment anti-CD40L only partially inhibited the B cell expansion resulted from anti-CD28 treatment.

More recent evidence indicates an expansion of the role of the CD40/CD40L in cellular interactions beyond Ab formation. Besides providing activating signals to B cells, CD40 also promotes B cell survival (20). It has been shown that activation of CD40 could inhibit anti-IgM-induced apoptosis in B cell lymphomas including WEHI231, Burkitt’s lymphoma, and Ramos, and in activated B cells. In contrast, in some lymphomas, ligation of CD40 could enhance the expression of Fas (46) and the susceptibility to Fas-induced apoptosis (47). Therefore, CD40-mediated protection of B cells from apoptosis is independent of the Fas pathway. However, most of the information concerning the role of CD40/CD40L in the regulation of apoptosis was derived from the in vitro system in combination with the signals from the B cell Ag receptor. The direct signals from CD40 are not clear. Nevertheless, it has been shown that CD40 ligation could induce the expression of Bcl-X (48). Recent studies have shown that CD40/CD40L interaction is also required for the formation of B memory cells and germinal centers, and signaling through CD40 prevents apoptosis of germinal center B cells. The increase in B cell number induced by repeated injection of CD28 is likely to be due to the induction of the expression of CD40L. The B cells from these mice showed prolonged survival in in vitro culture and resistance to anti-IgM- and FasL-induced apoptosis. Our data also show that the increase of B cells is due to the increase in B cell survival and cell proliferation (Fig. 7). This increase in cell survival may switch the balance between "life and death" toward more survival. Although, we have shown that the B cells bear the morphology of blasts, there is no significant increase in the amount of serum Ig- or hamster Ig-specific Abs. Therefore, CD28-regulated CD40L expression could play a critical in the maintenance of B cell homeostasis.

View larger version (12K): [in this window] [in a new window]   FIGURE 7. Proliferation of splenocytes from mice treated with anti-CD28 in the presence or absence of LPS. Mice were treated with 200 µg anti-CD28, 20 µg LPS, or both, with three doses at 48-h intervals. Mice were euthanized, and splenocytes were isolated and cultured in vitro for 6 h. Cell proliferation was measured by incubation with [3H]thymidine.

	Acknowledgments

 
We thank Drs. David W. Scott, Xiaorui Yao, and Achsah D. Keegan for critical discussions. We thank Dr. James Allison for the anti-CD28 B cell hybridoma, and Dr. Randolph Noelle for the anti-CD40L B cell hybridoma. We thank Ms. Elizabeth Smith and Ms. Yamei Gao for preparing histological sections. This is publication number 43 of the Department of Immunology, Holland Laboratory.

	Footnotes

 
¹ This work was supported by Research Grant 4426 of the Council of Tobacco Research-USA, INC., National Institutes of Health Grant AI43384-01, and the Biomedical Services of the American Red Cross. D.L.Y. is a recipient of a postdoctoral fellowship from the International Agency for Research on Cancer.

² Current address: Institut de Genetique et de Biologie Moleculaire et Cellulaire (CNRS/INSERM/ULP), 1 rue Laurent Fries, 67404 Illkirch, Communante Urbain de Strasbourg, France.

³ Address correspondence and reprint requests to Dr. Yufang Shi, Department of Immunology, Jerome H. Holland Laboratory of the American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. E-mail address:

⁴ Abbreviations used in this paper: CD40L, CD40 ligand; FasL, Fas ligand.

Received for publication August 28, 1998. Accepted for publication August 3, 1999.

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