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The Journal of Immunology, 2001, 167: 4059-4066.
Copyright © 2001 by The American Association of Immunologists

Soluble CD137 (4-1BB) Ligand Is Released Following Leukocyte Activation and Is Found in Sera of Patients with Hematological Malignancies1

Helmut R. Salih*, Helga M. Schmetzer{dagger}, Christine Burke*, Gary C. Starling*, Robert Dunn*, Renate Pelka-Fleischer{dagger}, Volkmar Nuessler{dagger} and Peter A. Kiener2,*

* Department of Immunology, Inflammation, and Pulmonary Diseases, Bristol-Myers Squibb, Pharmaceutical Research Institute, Princeton, NJ 08540; and {dagger} Med. Klinik III, Klinikum Grosshadern, Ludwig-Maximiliam University, Muenchen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of CD137 ligand (4-1BBL), a member of the TNF family of proteins, has been reported on several types of APCs, various carcinoma cells, and can be induced on activated T cells. In this study, we report that the soluble ligand was released constitutively at low levels from leukocytes and at higher levels following cellular activation. Release from cells was blocked by addition of a metalloproteinase inhibitor which concomitantly caused the accumulation of 4-1BBL on the cell surface. In addition, we show that a soluble form of 4-1BBL was present at high levels in the sera of some patients with various hematological diseases, but only at low levels in healthy donors. Soluble 4-1BBL was active in that it competed with recombinant 4-1BBL for binding to the 4-1BB receptor and was able to costimulate IL-2 and IFN-{gamma} release from peripheral T cells. These results indicate that the release of soluble 4-1BBL from the cell surface is mediated by one or more sheddases and likely regulates 4-1BB-4-1BBL interactions between cells in vivo. Cleavage of 4-1BBL to an active soluble form would alter both proximal and distal cellular responses, including cell survival and costimulatory or inflammatory responses, that are mediated through the 4-1BB pathway. This, in turn, would likely alter disease progression or outcome.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The 4-1BB molecule is a member of the TNF receptor gene superfamily, which includes an increasing number of identified proteins that are involved in the regulation of cell proliferation, differentiation, and programmed cell death (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). The natural ligand of 4-1BB (4-1BBL)3 is a type II integral membrane protein that is expressed on several types of APCs, such as activated B cells, monocytes, and splenic dendritic cells, and can be induced on T lymphocytes (12, 13, 14, 15, 16). In addition, 4-1BBL is expressed on tumor cells of lymphoid or myeloid origin and on various carcinoma cells (17). The interaction of 4-1BB with 4-1BBL+ APCs stimulates cell proliferation and production of IL-2 and IL-4 by CD4 T cells, and its role in T cell costimulation is most apparent in the absence of a strong B7-CD28 interaction (16, 18, 19).

Several of the members of the TNF ligand/receptor families signal in both directions, both through the respective receptor and back into the cells that express the ligand. Reverse signaling following cross-linking of 4-1BBL in lymphocytes results in the inhibition of proliferation, an increase in expression of Fas (CD95), and the induction of apoptosis (20). In carcinoma cells and macrophages, signaling though 4-1BBL stimulates the release of IL-8 (17, 21). A number of the ligands of the TNF superfamily are shed from the cell surface due to the activity of matrix metalloproteinases (MMP) or sheddases. For example, removal of Fas ligand (FasL) from the cell surface is thought to reduce its biological activity by diminishing its ability to cross-link Fas (24, 25), while cleavage of TNF and TNF-related activation-induced cytokine from the cell surface results in the release of active molecules that induce responses at sites distal to the producing cells (22, 23, 26).

In this study, we have used mAb raised against 4-1BBL to follow the levels of 4-1BBL surface expression and to detect the release of soluble 4-1BBL (s4-1BBL). We demonstrate that human 4-1BBL is released from lymphocytic and monocytic cells, notably upon activation of the cells, and this can be blocked by a MMP inhibitor (MMPI). The released s4-1BBL is functionally active in that it binds to 4-1BB and activates T cells. In addition, we report the presence of high levels of s4-1BBL in the sera of patients with various hematological malignancies, but not in those of healthy patients.


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

Raji cells, Daudi cells, and THP-1 cells were cultured in 10% FCS-RPMI 1640. T cells, B cells, and monocytes were isolated from healthy donors by density gradient sedimentation, rosetting, and elutriation (27). For the detection of s4-1BBL in culture supernatants, cells were cultured in 1% FCS-RPMI 1640 for the indicated times. Where indicated, monocytes in FCS-RPMI 1640 (1 x 106/ml) were incubated for 5 min with the MMPI before stimulation with 100 ng/ml LPS. After 7 h, the supernatants were removed and assayed for cytokines by ELISA (27)

Reagents

Anti-mouse {kappa}-FITC, anti-mouse IgG-PE, and anti-mouse HRP were purchased from Southern Biotechnology Associates (Birmingham, AL). The polyclonal anti-4-1BBL C20 Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The streptavidin-HRP was purchased from Vector Laboratories (Burlingame, CA); anti-mouse CD8 (53.6), anti-human CD3 (G19.4), anti-GST, anti-CD40 (G28.5), and the MMPI (28) were obtained from Bristol-Myers Squibb (Princeton, NJ). All fusion proteins were constructed and prepared as previously described (29, 30, 31).

Anti-4-1BBL mAb

mAb were raised to 4-1BBL (purified recombinant muCD8-hu4-1BBL, extracellular domain) by standard procedures using BALB/c mice (32). The mAb did not recognize mouse CD8 or other CD8 fusion proteins (CD154 and CD178); the selected mAb were purified from the culture supernatants by affinity chromatography on protein A-Sepharose. The two mAbs used in this work, clone 18 and clone 28, were of isotypes IgG1 and IgG2a, respectively; anti-GST mAbs of matching isotypes were used as mAb controls.

Flow cytometry

Cells were incubated with anti-4-1BBL mAb 18 or mouse IgG1 at 10 µg/ml and then with anti-mouse {kappa}-FITC (1:100) as secondary reagent. With monocytes, anti-mouse PE was used; FITC-stained cells were counterstained with propidium iodide for dead cell exclusion. Samples were analyzed on a FACScan (BD Biosciences, Mountain View, CA).

ELISA

To determine the capacity of the mAb to block the binding of 4-1BBL to its receptor (4-1BBIg), plates were coated with 10 µg/ml Ab anti-muCD8 overnight, washed, and incubated for 2 h at 37°C with 100 µl/well of a solution of 500 ng/ml CD8-4-1BBL in 10% FCS-PBS. Plates were blocked by addition of 100 µl of 15% BSA for 2 h at 37°C and then washed and incubated with 100 µl/well of the different clones of anti-4-1BBL mAb or control anti-GST mAb at 10 µg/ml in 10% FCS-PBS for 30 min before the addition of 100 µl/well of the 4-1BBIg fusion protein at 20 µg/ml in 10% FCS-PBS. Plates were incubated for 2 h at 37°C, then washed before the addition of 100 µl/well of a 1/5000 solution of donkey anti-human IgG-HRP in 10% FCS-PBS. The plates were further incubated for 1 h at 37°C, washed, and then developed using the TMB Peroxidase Substrate System (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The absorbance was measured at 450 nm (reference 725 nm).

For the detection of s4-1BBL using two anti-4-1BBL mAb that recognized different epitopes, plates were coated overnight at 4°C with 100 µl/well of the capture anti-4-1BBL mAb 18 at 2 µg/ml in PBS. The plates were blocked and washed as before and the standards (recombinant CD8-4-1BBL) and samples were then added and the plates incubated overnight at 4°C. For analysis of patient samples, sera were diluted 1/3 in 5% BSA, clarified by centrifugation at 14,000 rpm (15 m) before addition to the plates. After incubation, plates were washed, 100 µl of biotinylated anti-4-1BBL mAb 28 at 2 µg/ml in 10% FCS-PBS was added for 2 h at 37°C. The plates were then washed before the addition of streptavidin-HRP (1/2000 solution in 10% FCS-PBS) for 1 h at 37°C, and subsequently washed and developed as described above; 4-1BBL concentrations in supernatants are expressed as mean ± SEM of quadruplicate samples.

To determine the ability of s4-1BBL released from cells to bind to 4-1BBIg in a competitive ELISA, plates were coated with 10 µg/ml 4-1BBIg overnight at 4°C and then blocked and washed as above. In a separate plate, 120 µl of a 250 pg/ml solution of CD8-4-1BBL in 10% FCS-PBS was dispensed into each well. To the first row, 120 µl/well concentrated culture supernatant (at 250 pg/ml s4-1BBL) from PMA-stimulated Raji cells containing 250 pg/ml CD8-4-1BBL was added and the rows were then serially diluted 2-fold; 100 µl from each well was then transferred to the 4-1BBIg-coated plate and incubated for 2 h at 37°C. After washing, 100 µl/well of a solution of 2 µg/ml anti-muCD8 was added to detect bound CD8-4-1BBL. Plates were incubated for 2 h at 37°C, washed, and 100 µl/well of a 1/5000 solution of donkey anti-rat IgG-HRP was added; the plates were incubated for 1 h at 37°C and developed as described above.

SDS-PAGE and Western blot analysis

Raji cell lysates (5 x 107 cells/ml in 1% Triton X-100 lysate buffer (17, 27)), Raji cell supernatants (1 ml of supernatant, concentrated ~4-fold), or serum samples (1 ml) were immunoprecipitated with either a mixture of 2.5 µg mAb 18 and 28 (Raji samples) followed by protein G-agarose or 5 µg biotinylated anti-4-1BBL, 28 (serum samples), followed by streptavidin-agarose for 4 h. The immunoprecipitates were washed five times with PBS containing 0.1% Triton X-100 and 100 µM PMSF; the pellets were resuspended in 100 µl of SDS sample buffer; and the samples were separated on 4–12% tricine SDS-PAGE gels. The gels were blotted to polyvinylidene difluoride membranes, blocked with PBS containing 10% nonfat dried milk and 5% BSA, and then analyzed with 3 µg/ml goat anti-4-1BBL Ab. The membranes were the visualized with a HRP-labeled rabbit anti-goat IgG Ab HRP-goat and chemiluminescence reagent (NEN Life Science Products, Boston, MA).

T cell assays

Anti-CD3 (100 ng/ml) was immobilized on the surface of 96-well plates, the plates were washed, and then various amounts of a 10-fold concentrated culture supernatant of Raji cells (derived from 2 x 106 cells/ml in 1% FCS-RPMI 1640 for 24 h) diluted to 150 µl/well, either alone or with the addition of 10 µg/ml neutralizing anti-4-1BBL mAb 28 or the control anti-GST mAb, was added. Alternatively, fresh culture medium (no cells) that had been concentrated was used. The plates were kept for 30 min at room temperature before the addition of 1 x 105 peripheral T cells in 100 µl of 10% FCS-RPMI 1640 per well. The T cells were incubated at 37°C for 48 h before removal of culture supernatant and assayed for the release of IL-2 and IFN-{gamma} by ELISA according to the manufacturer’s instructions (OptEIA; BD PharMingen, San Diego, CA). Cytokine concentrations in supernatants are expressed as mean ± SEM of six replicates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Monoclonal Ab testing and development of the s4-1BBL ELISA

Two different clones of mAb raised to recombinant 4-1BBL were chosen for use in this study; both mAb bound to 4-1BBL expressed on the surface of Raji cells (Fig. 1GoA). Preincubation of the cells for 30 min with 20 µg/ml 4-1BBIg but not 20 µg/ml B7-2Ig before the addition of 10 µg/ml mAb reduced the binding of both anti-4-1BBL Ab clones 18 and 28, with a more marked effect seen on the binding of mAb 28 (Fig. 1GoA). The staining with mAb 18 in the presence of B7-2Ig gave the highest signal compared with the mouse isotype control, hence this was chosen for further FACS experiments to follow expression on the surface of cells.



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  		FIGURE 1. mAb characterization. A, Surface staining: Raji cells were incubated for 30 min in the presence of either 4-1BBIg or B7-2Ig before the addition of anti-4-1BBL mAb or the respective isotype control. Cells were counterstained with propidium iodide. Shaded histograms, 4-1BBL staining after preincubation with the B7-2Ig; open histograms, after preincubation with the 4-1BBIg; dotted lines, control mouse IgG1. B and C, Effect of mAb on receptor-ligand binding. B, CD8-4-1BBL was immobilized on 96-well plates and incubated with the different clones of anti-4-1BBL mAb or control anti-GST mAb (both at 10 µg/ml) before the addition of 10 µg/ml 4-1BBIg; C as in B but with a titration of anti-4-1BBL or GST mAb. The human IgG tail of the 4-1BBIg was then detected using anti-human IgG-HRP. Results in B are shown as mean of quadruplicates representing percentage of the control. Results in C are from eight replicates for each sample. E–G, Sensitivity and selectivity of ELISA for 4-1BBL: 96-well plates were coated with mAb #18; E, CD8-4-1BBL was added in serial dilution; or F, CD8-4-1BBL, CD8-FasL, or CD8-CD40L at 1 µg/ml were added and then subsequently detected with biotinylated mAb #28 followed by streptavidin-HRP. G, Alternatively, plates containing immobilized CD8-4-1BBL were developed using biotinylated mAbs (2 µg/ml) to 4-1BBL, CD3, or FasL. The mean and SD of eight replicates are shown.

 
The ability of the mAb to block the interaction of 4-1BBL with the 4-1BB receptor was then evaluated by ELISA. The anti-4-1BBL mAb or a control anti-GST mAb were incubated with immobilized CD8-4-1BBL for 10 min before the addition of the 4-1BBIg fusion protein. Both mAb 18 and 28 markedly reduced binding of 4-1BBL to its receptor in a dose-dependent manner with mAb 28 being more effective; the isotype-matched anti-GST mAb had no effect.(Fig. 1Go, B and C). This confirmed the results obtained in the competition FACS experiment. The anti-4-1BBL mAb 18 and 28 recognized different epitopes on 4-1BBL and did not cross-compete with each other for Ag (data not shown), and therefore these were used to establish an ELISA for s4-1BBL. The standard curve using the recombinant CD8-4-1BBL indicated that the sensitivity was down to ~2 pg/ml (Fig. 1GoD). The specificity of the mAb in the ELISA was further evaluated. The mAb did not recognize high levels of soluble recombinant CD40L or FasL (Fig. 1GoF), and the assay was dependent on the dose of the second mAb (Fig .1E). Additionally, irrelevant second mAbs (biotinylated anti-GST, anti-CD3, or biotinylated anti-FasL) gave no signal in the ELISA (Fig. 1GoG).

Regulation of expression and cleavage of membrane-bound 4-1BBL

Raji, THP-1 cells, or peripheral monocytes (5 x 105 cells) were cultured for 24 h in 24-well plates in 2 ml FCS-RPMI 1640 in the presence or absence of activating agents, and the levels of surface expression of 4-1BBL were then determined by FACS using the anti-4-1BBL mAb 18 (Fig. 2Go). Treatment of the Raji cells with 25 ng/ml PMA (Fig. 2GoA1) or with 5 µg/ml of both anti-IgM Ab and anti-CD40 mAb (both at 5 µg/ml; Fig. 2GoB1) increased the constitutive surface expression of 4-1BBL (~2.3- and 2.7-fold, respectively) compared with untreated cells. Addition of 10 µM MMPI to the culture medium further increased the levels of membrane-bound 4-1BBL, both in PMA- (Fig. 2GoC1) or Ab-stimulated (Fig. 2GoD1) cells (~6- and 4-fold, respectively, compared with levels of expression in untreated cells) and in untreated cells (Fig. 2GoE1; ~1.7-fold).



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  		FIGURE 2. FACS analysis of the modulation of 4-1BBL surface expression and cleavage. Cells were cultured in the presence or absence of the indicated compounds. After 24 h, cells were stained for 4-1BBL surface expression. Open histograms, 4-1BBL expression on untreated cells; shaded histograms, 4-1BBL expression on cells after treatment; dotted lines, staining with the isotype control. Column 1, Raji cells; column 2, THP-1 cells; column 3, peripheral blood monocytes. Cells were treated as indicated in the panels and as described in Materials and Methods

 
The monocyte cell line THP-1 showed only low levels of constitutive cell surface 4-1BBL expression (Fig. 2Go), and this increased slightly (~1.5-fold) upon treatment with 50 ng/ml PMA (Fig. 2GoA2) or 50 ng/ml PMA along with 1 µg/ml ionomycin (Fig. 2GoB2). Addition of MMPI at 10 µM slightly increased the levels of 4-1BBL expression in both PMA (Fig. 2GoC2) and PMA with ionomycin (Fig. 2GoD2)-treated cells (~2-fold compared with untreated); in contrast, addition of MMPI to untreated cells did not result in a detectable change (Fig. 2GoE2).

Similarly, only low levels of 4-1BBL surface expression were detected in peripheral monocytes (Fig. 2Go); the expression increased significantly following addition of 50 ng/ml PMA (Fig. 2GoA3) or 50 ng/ml PMA in combination with 1 µg/ml ionomycin (Fig. 2GoB3) (~2.3- and 3-fold increase in the mean, respectively). In addition, treatment with LPS resulted in a minor up-regulation of 4-1BBL expression, whereas addition of IFN-{gamma} to the culture medium had no effect (data not shown). As was seen with THP-1 cells, addition of 10 µM MMPI further increased the levels of 4-1BBL expressed on the cell surface following treatments (Fig. 2GoC3: with PMA; D3: with PMA and ionomycin) but only to a minor extent (~3-fold for PMA and 3.4-fold increase with PMA and ionomycin, respectively, over untreated cells); again little effect (~1.2-fold) was seen by addition of MMPI without additional treatments (Fig. 2GoE3). Investigation of peripheral B and T cells revealed only minor changes in the levels of 4-1BBL surface expression, whereas analysis of Daudi cells revealed that 4-1BBL was not constitutively expressed and that there was no significant effect upon treatment of the cells (data not shown).

Regulation of release of s4-1BBL in vitro

It has been shown that the ligands from several members of this family are cleaved from the surface of cells and released as soluble proteins. Since treatment of cells with MMPI increased cell surface expression of 4-1BBL, we investigated by ELISA whether there was release of s4-1BBL from cells and cell lines. We cultured 2 x 106 (Raji) or 4 x 106 (others) cells/ml for 24 h in 24-well plates in RPMI 1640 containing 1% FCS and determined the levels of s4-1BBL released into the supernatants. In the absence of any stimulation, Raji cells released significant levels of s4-1BBL (Fig. 3GoA). Addition of a MMPI to untreated cells caused a reduction of detectable levels of s4-1BBL but this just failed to reach statistical significance (p = 0.07). Treatment of cells with 25 ng/ml PMA or 5 µg/ml of both anti-IgM Ab and anti-CD40 mAb for 24 h markedly increased the release of s4-1BBL compared with untreated control cells (both p < 0.01); PMA treatment was more effective than the stimulation with the Abs. Addition of MMPI significantly reduced the stimulation-induced release of s4-1BBL from both PMA- and Ab-treated cells (p < 0.01). After 48 h of stimulation, the levels of spontaneously released s4-1BBL in Raji cell supernatants were lower than those after 24 h and only PMA treatment raised highly elevated levels of s4-1BBL (data not shown).



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  		FIGURE 3. ELISA for regulation of release of s4-1BBL, effect of MMPI. Cells were incubated for 24 h in the presence of the indicated compounds. After incubation, supernatants were harvested and investigated by ELISA. The data shown are means of quadruplicates with SDs of one representative experiment from a total of four. A, Raji cells; B, THP-1 cells; C, peripheral B cells; D, peripheral T cells. E, Effect of the MMPI inhibitor (10 µM) on cytokine release from peripheral monocytes. Cells were incubated with the MMPI for 5 min before stimulation with 100 ng/ml LPS. {square}, TNF-{alpha}; {blacksquare}, IL-1{beta}. Samples were assayed as eight replicates. The data are from one experiment representative of a total of three.

 
In the monocytic cell line THP-1, the levels of spontaneous release of 4-1BBL were low (<2 pg/ml), mirroring the low level of expression on the cell surface (Fig. 3GoB). Addition of 50 ng/ml PMA or 50 ng/ml PMA along with 1 µg/ml ionomycin significantly (p < 0.01) increased the levels of s4-1BBL released after 24 h; PMA combined with ionomycin was most effective. Again, addition of 10 µM MMPI before treatment significantly (p < 0.01) reduced the levels of s4-1BBL released from stimulated cells. In Daudi cells, which in a previous report have been shown to express cell surface 4-1BBL (33), it was not possible to detect s4-1BBL in the 24-h culture supernatants; after 48 h, very low levels could be detected and addition of 50 ng/ml PMA caused a slight increase (data not shown). The release data were consistent with our finding that there was no or very low cell surface expression on the Daudi cells.

Investigation of culture medium derived from peripheral B cells of healthy donors showed that, without subsequent treatment of the cells, levels of s4-1BBL were close to the lower detection limit of the ELISA (Fig. 3GoC). Stimulation of the cells for 24 h with 50 ng/ml PMA or 5 µg/ml of both anti-IgM Ab and anti-CD40 mAb induced a significant increase (both p > 0.01) of s4-1BBL; again, addition of 10 µM MMPI before treatment significantly reduced the activation-induced release (p < 0.01). Analysis of the culture supernatants from peripheral T cells from healthy donors revealed no spontaneous release and no significant induction of release of s4-1BBL following stimulation with 50 ng/ml PMA (Fig. 3GoD). However, treatment with 50 ng/ml PMA in combination with 1 µg/ml ionomycin induced release of significant (p < 0.01) levels of s4-1BBL, and this was blocked (p < 0.01) by the addition of 10 µM MMPI to the culture.

The MMPI used in these experiments has been reported previously (28). To confirm its activity, the effect of the inhibitor on LPS-stimulated release of TNF-{alpha} and IL-1{beta} from peripheral monocytes was determined. At 10 µM, the MMPI effectively blocked the release of TNF from the monocytes (Fig. 3GoE) with a concomitant increase in cell surface expression (data not shown); however, the MMPI had no effect on the release of IL-1{beta}. This suggest that the MMPI inhibits a sheddase-like activity that is responsible for the release of TNF and s4-1BBL.

Influence of s4-1BBL on 4-1BB receptor-ligand interaction

To characterize the functional activity of the released s4-1BBL, we determined whether the s4-1BBL was able to interfere with the binding of recombinant 4-1BBL (CD8-4-1BBL) to 4-1BBIg in a competitive ELISA. The supernatant of PMA-stimulated Raji cells or the unconditioned medium control were concentrated about 10-fold and then evaluated for their ability to compete with recombinant CD8-4-1BBL for binding to 4-1BBIg. The s4-1BBL from the Raji culture supernatants reduced the binding of recombinant 4-1BBL to 4-1BBIg in a dose-dependent manner (Fig. 4GoA); the concentrated medium (with no cellular conditioning) had no effect. Other soluble CD8 fusion proteins, CD40L and FasL, at 1 µg/ml had no effect on the binding of s4-1BBL to 4-1BBIg (Fig. 4GoB). This indicates that the s4-1BBL released from cells was able to bind to the 4-1BB receptor and specifically compete with the recombinant 4-1BBL for binding. Using 250 pg/ml of the recombinant 4-1BBL in the assays, the IC50 for s4-1BBL was ~100 pg/ml. With adjustment for the Mr of CD8-4-1BBL and s4-1BBL (46,000 and 21,000, respectively), this suggests that, within the limits of experiments, both the recombinant and the s4-1BBL bound with similar affinity to the 4-1BB receptor.



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  		FIGURE 4. Influence of s4-1BBL on 4-1BB receptor-ligand interaction. A, The ability of s4-1BBL to block binding of CD8-4-1BBL to immobilized 4-1BBIg was measured in a competitive ELISA with fixed a fixed concentration of CD8-4-1BBL and serial dilutions (1/1) of s4-1BBL. B as in A except that a fixed concentration of s4-1BBL (700 pg/ml), recombinant soluble FasL (1 µg/ml), or recombinant soluble CD40L (1 µg/ml) was used to compete with binding of CD8-4-1BBL (5 ng/ml) to immobilized 4-1BBIg. Binding of CD8-4-1BBL was detected using rat anti-mouse CD8 mAb followed by anti-rat IgG-HRP. The data shown are means of quadruplicates with SDs of one representative experiment from a total of three.

 
Western blot analysis of s4-1BBL

To determine the nature of the released s4-1BBL, the ligand was immunoprecipitated from Raji cell lysates, Raji cell supernatants, and patient serum samples and analyzed by Western blot using a polyclonal Ab (to the C-terminal region) to detect the protein fragments (Fig. 5Go). Lysates from intact cells showed a major band at ~32 kDa with a smaller fragment at ~26 kDa (Fig. 5Go, lane 1). In the serum samples from two different patients, a doublet was observed around 26 kDa with a smaller band at ~10 kDa (Fig. 5Go, lanes 2 and 3). In the immunoprecipitates of supernatants of Raji cells, bands at ~26 and 19 kDa could be detected (Fig. 5Go, lanes 4 and 5). Immunoprecipitation with anti-GST did not bring down protein that was recognized by the anti-4-1BBL Ab (Fig. 5Go, lane 6). The detection of several bands attributable to s4-1BBL forms in these samples suggests that there may be several different processing steps. Alternatively, despite the presence of protease inhibitors, it is possible that there was degradation during the sample preparation.



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  		FIGURE 5. Western blot analysis of s4-1BBL samples. s4-1BBL was immunoprecipitated from Raji cell lysates, Raji cell supernatants, and patient serum samples, separated by SDS-PAGE, and then analyzed by Western blot using a polyclonal anti-4-1BBL (C terminus reactive). Lane 1, Raji cell lysates; lanes 2 and 3, patient sera; lanes 4 and 5, supernatants from Raji cells treated with PMA; and lane 6, anti-GST immunoprecipitate from Raji cell supernatants. The data shown are from one experiment representative of a total of two.

 
s4-1BBL can stimulate T cell cytokine production

To determine whether the s4-1BBL was biologically active, we assessed the ability of cell supernatants of Raji cells (without PMA treatment) to costimulate T cells. T cells alone or stimulated with low levels of anti-CD3 raised very little release of IL-2 or IFN-{gamma}. The addition of concentrated Raji cell culture medium containing a final concentration of 150 pg/ml s4-1BBL (by ELISA) induced activation of T cells as determined by the release of both cytokines (Fig. 6Go, A and B). Incubation of the concentrated s4-1BBL-containing supernatant with 10 µg/ml of the neutralizing anti-4-1BBL mAb 28 for 30 min at room temperature before addition of T cells markedly reduced the levels of IL-2 and, to a lesser extent, of IFN-{gamma} (both p < 0.01), whereas incubation with a control mAb (anti-GST) of the same isotype had no effect. Furthermore, the stimulation of both IL-2 and IFN-{gamma} from the cell supernatants by s4-1BBL was dose dependent (Fig. 6GoC). This indicates that the observed T cell activation by the Raji cell supernatant was, at least in a significant part, due to s4-1BBL.



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  		FIGURE 6. Activation of T cells. Determination of the effect of Raji cell supernatants on anti-CD3-induced cytokine production by human peripheral T cells in the absence or presence of anti-4-1BBL (mAb 28) or anti-GST mAbs. A, IL-2; B, IFN-{gamma}; C, dose response of T cells to supernatants (dialyzed and concentrated) from Raji cells. The amount of s4-1BBL in the supernatants was determined by ELISA as described in Materials and Methods. Cytokine concentrations in supernatants are expressed as mean and SD of six replicates.

 
s4-1BBL is detected at high levels in sera from leukemia patients

The results obtained in vitro indicated that s4-1BBL could be released from cells in culture. To determine whether s4-1BBL release was evident in the serum of healthy volunteers or patients with various hematological malignancies, serum samples were collected and assayed by ELISA for s4-1BBL. The sera of the healthy volunteers contained low levels of s4-1BBL (Fig. 7Go). The ELISA revealed levels of s4-1BBL between 1.3 and 5.3 pg/ml, with mean and median values of 3.2 and 3 pg/ml, respectively. Sera from patients with non-Hodgkin lymphoma (NHL) showed levels of s4-1BBL ranging between 1.2 and 615 pg/ml, with a mean of 57 pg/ml and a median of 30.5 pg/ml. In patients with myelodysplastic syndrome (MDS), the range of s4-1BBL was between 1 and 3280 pg/ml, the mean being 210 pg/ml and the median 32 pg/ml. The highest levels of s4-1BBL were detected in sera from patients with a variety of subclasses of acute myeloic leukemia (AML); the range was between 1.5 and 5450 pg/ml, with a mean of 682 pg/ml and a median of 53 pg/ml. The two sera of patients with chronic lymphatic leukemia revealed s4-1BBL at concentrations of 3.6 and 10.4 pg/ml.



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  		FIGURE 7. Levels of s4-1BBL in sera of patients and healthy donors. Serum samples were investigated by ELISA. The data shown are means of quadruplicates. CLL, chronic lymphatic leukemia. n, number of donors in each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
4-1BB has been shown to have an important role in costimulatory pathways in T cells. However, recent reports indicate that, like other members of the TNF/TNFR family, the 4-1BB/4-1BBL system may have more diverse roles than originally described. Although 4-1BB expression is mainly restricted to T cells, its ligand is expressed on a variety of cell types. In addition to the forward signaling via the 4-1BB receptor, reverse signaling occurs through 4-1BBL (for review, see 34). cross-linking of 4-1BBL with 4-1BB-expressing insect cells induced B cell proliferation synergistically with anti-IgM Abs (15), and a 4-1BB-Fc fusion protein activated monocytes after reverse signaling through 4-1BBL. This increases the expression of IL-6, IL-8, TNF-{alpha}, and ICAM expression and decreases that of Fc{gamma}RIII (21). Furthermore, transduction of signals through 4-1BBL leads to the production of M-CSF that functions as survival factor for monocytes (35) and mediates monocyte proliferation (36). In T cells, reverse signaling through 4-1BBL inhibits proliferation induced by anti-CD3 (37), induces apoptosis independently from Fas in resting T lymphocytes, and up-regulates expression of CD95 (Fas) on lymphocytes (20). Furthermore, T lymphocytes from 4-1BB knockout mice exhibit an elevated capacity to proliferate in response to anti-CD3 or mitogens, and this effect was abrogated by triggering 4-1BBL with insect cells overexpressing the 4-1BB receptor (15). On carcinoma cells, 4-1BBL is constitutively expressed and reverse signaling induces release of IL-8 by these cells (17). Taken together, these findings indicate that cells that express 4-1BBL both stimulate responses in 4-1BB-expressing cells and also, through 4-1BBL reverse signaling, undergo a variety of responses that depend on the cell type. Thus, changes in the expression of cell surface 4-1BBL are likely to play an important role in regulating the outcome of cell-cell interactions.

Several ligands of the TNF family are released in a soluble form following cleavage from the cell surface. This may significantly affect cell-cell interactions and responses to ligand or may be responsible for provoking more distal responses to cellular activation (22, 23, 24, 25, 26). In this study, we have developed mAb that detect both cell surface and soluble forms of 4-1BBL. We show that expression of 4-1BBL was up-regulated on the surface of Raji cells 24 h after treatment with PMA or a combination of anti-IgM and anti-CD40. Addition of a MMPI increased the levels of 4-1BBL expressed on the cell surface of either constitutive or activated cells. In monocytic cells, both THP-1 cells and peripheral monocytes revealed a minor up-regulation of 4-1BBL expression after treatment with PMA or PMA in combination with ionomycin. Addition of MMPI caused a small, but reproducible further increase.

Release of s4-1BBL was observed from several cells, the most notable were Raji cells, which constitutively released s4-1BBL and this increased upon activation of the cells with PMA or anti-IgM and anti-CD40. Addition of MMPI reduced both the levels of constitutively released s4-1BBL and those released after stimulation of the cells. We also found that treatment of B cells with PMA or the Ab combination increased the release of s4-1BBL and this too was blocked by addition of MMPI. Similarly activation of T cells with PMA and ionomycin raised the release of s4-1BBL, which again was blocked by the presence of the MMPI. Treatment of THP-1 cells with PMA or PMA and ionomycin induced a significant increase in the levels of s4-1BBL and this was blocked by addition of the MMPI. In peripheral monocytes, it was difficult to detect changes in the levels of s4-1BBL; only very low levels of s4-1BBL were released constitutively and under the incubation conditions (low serum) the cells rapidly undergo apoptosis (27). This may have complicated detection of s4-1BBL in the ELISA. Overall, these results indicate that, in a variety of different cell types, s4-1BBL is released by cleavage from the cell surface and this can be inhibited by a MMPI. Several different lower molecular mass forms were observed which suggests that processing may be complex, involving one or more sheddase or MMP.

The consequences of release of 4-1BBL may be dependent on the cell type. Shedding of the ligand from the cell surface of T lymphocytes might decrease their sensitivity to apoptotic stimuli (20). In APCs such as B cells and monocytes, where signaling through 4-1BBL has been shown to take part in activation, proliferation, and costimulation (15, 21, 35, 36), shedding may function as a mechanism to limit inflammatory or costimulatory responses induced by the local cell-cell interaction. We found that s4-1BBL effectively competed with recombinant CD8-4-1BBL for the binding to 4-1BBIg and was able to deliver a costimulatory signal to peripheral T cells in the presence of immobilized anti-CD3. This costimulatory signal was blocked by a neutralizing anti-4-1BBL mAb, suggesting that binding of s4-1BBL to its receptor was largely responsible for the induction of the cytokines from T cells and, furthermore, that the s4-1BBL was functionally active. As reported previously (17), it was not possible to see stimulation of IFN-{gamma} in the absence of anti-CD3.

We analyzed serum samples from healthy donors and patients with various malignant hematological diseases and found that, while only very low levels of s4-1BBL were present in sera from healthy donors, elevated levels were detectable in many of the sera of patients with AML, MDS, and NHL (statistical significance: p = 0.001, 0.014, and 0.03 compared with healthy patients for AML, MDS, and NHL, respectively). In several patients, the levels of s4-1BBL in the sera were markedly higher than those found necessary in vitro to effectively stimulate T cells; this indicates that the in vivo release from cells and/or dispersion to a distal site is likely to have physiological significance. Release of 4-1BBL from leukemic cells may provide a mechanism for the cells to escape local immune surveillance by limiting costimulation of the host lymphocytes and by reducing apoptotic signals through 4-1BBL back into the tumor cell. In addition, release of active s4-1BBL to distal sites may be responsible for some of the pathophysiology of the disease.

Overall, the impact of cleavage of membrane 4-1BBL to its soluble form on cell-cell interactions and its role in the pathological mechanism of hematological diseases requires further elucidation. In future studies, it will be important to determine whether the levels of s4-1BBL in patient sera at the time of diagnosis of leukemia, and during treatment, correlate to the progress and outcome of the disease. For example, are high levels of s4-1BBL indicative of progression from MDS to an acute leukemia? Furthermore, it remains to be determined whether modulation of 4-1BBL release or blocking of its cleavage by MMPI might be of benefit in the treatment of hematological or immunological diseases.


   Footnotes
 
1 This work was supported by a grant from Mildred-Scheel Stiftung fuer Krebsforschung, Germany (to H.R.S.). Back

2 Address correspondence and reprint requests to Dr. Peter A. Kiener, Department of Immunology and Inflammation, K14-04, Bristol-Myers Squibb, P.O. Box 4000, Princeton, NJ 08543. E-mail address: kienerp{at}bms.com Back

3 Abbreviations used in this paper: 4-1BBL, 4-1BB ligand; FasL, Fas ligand; MMP, matrix metalloproteinase; MMPI, matrix metalloproteinase inhibitor; AML, acute myeloic leukemia; NHL, non-Hodgkin lymphoma; MDS, myelodysplastic syndrome. Back

Received for publication February 21, 2001. Accepted for publication August 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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