Autocrine transforming growth factor- from chronic lymphocytic leukemia-β cells interferes with proliferative T cell signals

Immunobiol., vol. 200, pp. 128-139 (1999)

From the [Department of Medicine III, Johannes Gutenberg University, Mainz, and the 2Department of Medicine III, Technical University, Munich, Germany

Autocrine Transforming Growth Factor-p from Chronic Lymphocytic Leukemia-B Cells Interferes with Proliferative T cell Signals MARTIN SCHULER 1, THERESA TRETTER2, FOLKER SCHNELLER2, CHRISTOPH HUBER 1, and CHRISTIAN PESCHEL2

Received June 15, 1998 . Accepted in revised form October 21, 1998

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of noncycling B cells in lymphatic and extralymphatic tissues. In the present study we investigated the possible contribution of TGF-~, as secreted by CLL-B cells, on this low proliferative state. CLL-B cells were shown to express TGF-~ RNA and to release bioactive TGF-~ into culture supernatants. Antibody neutralization of endogenously secreted TGF-~ increased the proliferation of CLL-B cells as cultured in the presence of IL-2 or IL-4 or in direct contact with activated CD4+ T cells. In these culture systems, addition of exogenous TGF-~ downregulated basal and cytokineinduced proliferation of CLL-B cells. In contrast, neither neutralization of endogeneous TGF-~, nor addition of exogeneous TGF-~ changed the proliferation of CLL-B cells as cultured in the CD40 system. In order to further explore this differential antiproliferative effect of TGF-~, cytokine secretion of B cells and of CD4+ T cells as well as surface marker expression of CD4+ T cells were assessed in relation to TGF-~: There was no negative effect of TGF-~ on autocrine secretion of TNF-a or sCD23 by CLL-B cells. Unlike tonsillar B cells, CLL-B cells cultured alone or in the CD40 system did no release significant amounts of IL-6 or IL-8 into supernatants. Secretion of IL-2 or IL-4 by activated CD4+ T cells was higher, when T cells were cocultured with normal tonsillar B cells than with CLL-B cells. The amount of IL-2 or IL-4 released by CD4+ T cells cocultured in direct contact with tonsillar or CLL-B cells was not consistently influenced either by neutralization of endogenous TGF-~ or by addition of TGF-~. Exogenous TGF-~ did not downregulate expression of CD40L, CD27, CD28, CD54 or mTNFa by T helper cells activated with anti-CD3 or PHA. In conclusion, autocrine secretion of TGF-~ exhibits an antiproliferative effect on CLL-B cells. This effect is most relevant in B cells cultured in direct contact with activated CD4+ T cells suggesting an indirect mode of action.

Abbreviations: TGF-f3 = transforming growth factor-~; CLL = chronic lymphocytic leukemia; PBMNC = peripheral blood mononuclear cells; mAb = monoclonal antibody, FACS = flow cytometry; I L = interleukin; TNF = tumor necrosis factor; CD40L = CD40 ligand. °1999 by URBAN & FISCHER

0171-2985/99/200/01-128 $12.00/0

Amocrine TGF-~ in B-CLL .

129

Introduction The main histopathologic feature of CLL is a progressive expansion of predominantly noncycling clonal B cells in lymphatic and extralymphatic tissues, leading to lymphadenopathy, organomegaly, and suppression of normal hematopoiesis. In addition, immune dysfunction resulting in infections, and autoimmune phenomena is frequently encountered in CLL patients (1, 2). Impairment of programmed cell death may contribute to this characteristic accumulation of CLLB cells in vivo (3). In contrast, when cultured in vitro, CLL-B cells rapidly undergo apoptotic cell death (4), largely precluding useful in vitro study of the biology of CLL. Recently, this problem was overcome by the development of the CD40 system (5-7), which allows prolonged survival and even proliferation and differentiation of B cells in vitro. Moreover, when coculturing clonal B cells in direct contact with autologous or allogeneic T cells, their resting and unresponsive state could be further reversed (8-10). TGF-~ is a multifunctional cytokine, which is involved in regulation of cell growth and survival, immune functions, and wound healing (11-13). In mitogen-activated B cells, TGF-~l induces growth arrest in mid- to late G 1 phase by a mechanism involving inactivation of CDK2 by p27 Kip1 , and inhibition of Cyclin A expression (14). TGF-~ (at low levels) is capable of stimulating immunoglobulin (Ig) production in B cells, whereas at high levels TGF-~ acts inhibitory on Ig synthesis. Moreover, TGF-~ directs switch recombination towards the IgA isotype (15). Mice being disrupted of the TGF-~1 gene show a massive multifocal inflammatory disease leading to wasting and early death (16, 17). In vivo, the secretion of negative regulators such as TGF-~ by clonal B cells might contribute to the pathophysiology of CLL: CLL-B cells were shown to express and to release TGF-~ In vivo (18, 19), but variable responses were observed in vitro (19-21). In the present study, we investigated the effects of autocrine TGF-~ on B cells themselves, as well as on cocultured CD4+ T cells. We found that autocrine TGF-~ in CLL-B cells mainly impairs proliferative signals as provided by CD4+ T cells.

Materials and Methods Materials Recombinant human (rh) interleukin-2 (IL-2) was purchased from Hazleton. Rh IL-4 was obtained from ICC Inc. (Ismaning, Germany). Rh IL-IO (specific activity 5 x 105 U/mg) was purchased from Genzyme (Cambridge, MA, USA). Rh TGF-~l (specific activity 1.67-5 x 1Ql U/mg) was purchased from R&D Systems (Abingdon, UK). The rabbit anti-pan TGF-~ antibody AB-lOO-NA was obtained from R&D Systems. The murine mAb MAB89 binding CD40 was in part generously provided by Drs. S. SEALAND and J. BANCHEREAU (Dardilly, France), and in part purchased from Immunotech (Marseille, France). a 32 P-labeled nucleotides were purchased from Amersham Buchler (Braunschweig, Germany), and 3H-labeled thymidine was purchased from DuPont (Bad Homburg, Germany).

130 . M. SCHULER et al. Separation of B cells and CD4+ T cells

PBMNC were isolated from patients suffering from B-CLL after informed consent using centrifugation over Ficoll-Hypaque density gradient (Biochrom, Berlin, Germany). In all patients diagnosis was confirmed according to clinical and immunophenotypic criteria. The B cell fraction was isolated by negative selection of cells expressing CD2 and CD14 using immunomagnetic beads (Dynabeads M450, Dynal, Oslo, Norway) according to the manufacturer's instructions. Purity of the B cell fraction was >99% as confirmed by FACS analysis. For control experiments, the B cell fraction was isolated from fresh human tonsil tissue obtained at routine adenotomy. Purification of tonsillar B cell fraction was performed by negative selection as described for CLL-B cells. For CD4+ T cell cocultures PBMNC were isolated from normal volunteers by centrifugation over Ficoll-Hypaque (Biochrom). E Rosette negative (E-) and £+ cells were prepared by rosetting with neuraminidase-treated (Sigma, St. Louis, MO, USA) sheep red blood cells using standard procedures. Highly purified CD4+ cells were obtained by negative selection of cells expressing CD8, CDI4, CDI6, CDI9, or CD56. £+ cells were incubated with a cocktail of those mABs followed by depletion using sheep anti-mouse IgG coated magnetic beads (Dynabeads M450). This separation procedure was repeated once and resulted in a purity of CD4+ cells of >97%; contamination with monocytes and B cells was less than 1%. Cell cultures

For protein and RNA analysis, B cells were incubated in flasks (Nunc, Roskilde, Denmark) using 5 or 25 ml RPMI 1640 (Biochrom) supplemented with 10% fetal calf serum (FCS, Biochrom), penicillin 50 IU/ml, streptomycin 50 IU/ml, Na-pyruvate 1 mmollL, L-glutamine 2 mmollL, HEPES 10 mmollL, and modified Eagle's medium (MEM) non-essential amino acids xO.7 (Biochrom) as culture medium at 37°C and 5% CO 2, Cell concentration was 106/ml. For proliferation assays B cells were cultured at a concentration of 2.5 x 105 cells/ml in 96-well U bottom plates (Nunc) using 200 pI culture medium per well. In the CD40 system (5) B cells were additionally cocultured with murine Ltk-cells transfected with the Fey receptor CDw32 (kindly provided by Drs. S. SEALAND and J. BANCHEREAU, Dardilly, France), which were growth inhibited by y-irradiation with 5,000 rad, and the antibody MAB89. Ltk-cells were used at a Ltk-:B cell ratio of 1:10, and were allowed to adhere before addition of factors and B cells. The antibody MAB89 was used at a concentration of 500 pg/ml. For B cell-T helper cell cocultures 96-well flat bottom plates (Nunc) were coated with anti-CD3 (VIT3, 100 pg/well, kindly provided by Dr. W. HOLTER, Vienna, Austria) at 4 °C overnight was described elsewhere (10). Highly purified CD4+ cells, which were y-irradiated with 2,000 rad, were added at 105 cells/ml in a total volume of 100 pI culture medium. After 24 h B cells (2.5 x 105 cells/ml) and factors were added. TGF-~ bioassay

The mink lung cell line Mvl Lu (ATCC CCL-64) was obtained from ATCC (Rockville, MD, USA). Cells were grown in culture flasks with MEM Earle (Biochrom) supplemented with 10% FCS at 37°C and 5% CO 2 until confluent. Then, cells were trypsinized and transferred into 96-well flat bottom plates (Nunc) in MEM 1% FCS (200 pllwell) at a concentration of 2.5 x 10 5) cells/ml. The cell layer was allowed to become confluent for 24 h. Then, medium was replaced by culture supernatants to be assessed for TGF-~ bioactivity diluted in MEM FCS 1%. Following another incubation period of 24 h cells were labeled with 3H thymidine (DuPont) 1 pCi/well for 4 h, and cells were lysed and harvested on a PHD cell harvester (Cambridge Technologies Inc., Cambridge, MA, USA). Activity was measured by a Beckman LS 1801 liquid scintillation system (Beckman, Irvine, CA, USA). All experiments were done in triPlicate; concentrations of bioactive TGF-~ were calculated from a standard reference curve obtamed simultaneously.

Autocrine TGF-~ in B-CLL . 131 Northern blot analysis

Total cytoplasmatic RNA was isolated from B cells by the single step method of guanidiniuml phenol chloroform extraction as described previously (22). RNA was electrophorized on an 1% agarose-formaldehyde gel and transferred onto nylon membrane (Hybond-N, Amersham Buchler). Blots were hybridized to a 32 P-Iabeled eDNA probes using a random primer DNA labeling kit (Boehringer Mannheim, Germany). After washing they were exposed to Cronex-4-autoradiogaphy films (DuPont) at -70°C. The phTGFB2 construct (23, 24) was obtained from ATCC (Rockville, MD). The 2.138 kb EcoRI fragment containing the human TGF-~l eDNA was used for hybridization. Cell proliferation assays

B cells were cultured in triplicate in 96 well U-bottom plates, as described above, for 120 h. Then, cells were labeled with 3H thymidine (DuPont) 1 pCi/well for 16 h. For B cell-CD4+ T cell cocultures B cells were added to anti-CD3 activated CD4+ T cells and coincubated for 96 h. Labeling with 3H thymidine (DuPont) 1 pCi/well was performed for 16 h. Cells were lysed and harvested on a PHD cell harvester and activity was measured on a Beckman LS 1801 liquid scintillator. Enzyme-linked immunosorbent assays

TNF-a protein was measured by a commercially available ELISA kit (ELISA, Medgenix, Fleurs, Belgium). IL-2 and IL-8 protein were detected by Quantikine kits (R&D Systems, Minneapolis, MN, USA). IL-6 protein was measured with a Biotrak kit (Amersham, Little Chalfont, UK), and sCD23 protein was measured with an ELISA kit from T Cell Diagnostics Inc. (Woburn, MA, USA). IL-4 protein was measured by means of an ELISA using antibodies kindly provided by the Novartis Research Institute, Vienna, Austria, as described recently (25). Flow cytometry

Surface expression of CD40 ligand (CD154), CD54, CD27, membrane-bound TNF-a (mTNFa) and CD28 was detected by direct immunofluorescence using PE-labelled monoclonal antibodies (Pharmingen, Hamburg, Germany) on a Coulter Epics XL-MCL FACScanner. Statistics

Values obtained under various culture conditions were compared by means of the Friedman test. In a second step, multiple comparisons were performed by computing the Student-NewmanKeuls test.

Results Autocrine production of TGF-~ by CLL-B cells

CLL-B cells as well as normal tonsillar B cells expressed a 2.5 kB TGF-~l RNA fragment as detected by Northern blotting. No significant influence of various culture conditions on the level of TGF-~l RNA expression could be observed (data not shown). Secretion of bioactive TGF-~ by B cells isolated from 7 BCLL patients was assessed by the Mv1 Lu bioassay. Higher levels of bioactive TGF-~ were detectable upon stimulation in the CD40 system (Fig. 1). The

132 . M. SCHULER et al.

TGF-B release of normal tonsillar B cells followed the same range and distribution pattern (data not shown). Antiproliferative activity of autocrine

TGF-p on

CLL-B cells

Proliferation studies were performed with B cells from 14 B-CLL patients. B cells were incubated either alone (B cell culture), in presence of Ltk-cells expressing CDw32 and the antibody MAB89 (CD40 system), or in presence of irradiated CD4+ T cells, which were activated by the antibody VIT3 (B cellCD4+ T cell coculture). Increasing concentrations of the rabbit anti-pan TGF-~ antibody AB-100-NA led to an increment of 3H thymidine incorporation in CLL-B cells as stimulated by IL-2 (Fig. 2). The maximal effect was observed at

Medium IL-2 CD40 CD40 + IL-2

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50

100 150 200 250 300 350 TGF-B lpg/mil

Figure 1. Release of bioactive TGF-~ into culture supernatants (mean values) by B cells from 7 patients with CLL. Cells were cultured for 48 hours, and bioactive TGF-~ was measured by the Mvl Lu bioassay as described in Materials and Methods.

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Autocrine TGF-~ in B-CLL . 133

an antibody concentration of 20 pg/ml. For further experiments an excess antibody concentration of 50 pg/ml was used. In B cell culture, the low basal proliferation rate of CLL-B cells was significantly increased by IL-2, and IL-4 (data not shown). Neutralization of endogenous TGF-~ could further increase proliferation. Addition of exogenous TGF-~ had an antiproliferative effect on CLL-B cells as stimulated by IL-2 or IL-4 (Fig. 3A): In the CD40 system, the optimal B cell proliferation was obtained by stimulation with IL-2 plus IL-IO, as compared to stimulation with either IL-4 of IL2 alone (data not shown). Neutralization of endogenous TGF-~ did not significantly influence proliferation of CLL-B cells as stimulated by IL-2 plus IL-IO. Moreover, addition of exogeneous TGF-~ also had no significant antiproliferative effect (Fig. 3B). Maximal B cell proliferation was obtained in the B cell-CD4+ T cell coculture system by neutralizing endogenous TGF-~. Addition of exogenous TGF-~ decreased CLL-B cell proliferation below the basal level. When exogenous IL-2 or IL-4 (data not shown) was added in this system, basal CLL-B cell proliferation was further increased. However, this effect was significantly less pronounced as compared to neutralization of endogenous TGF-~ (Fig. 3C).

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134 . M.

SCHULER

et al.

Influence of TGF-~ on cytokine release from tonsillar and

CLl-B cells

The secretion of TNF-a, IL-6, IL-8, and sCD23 protein by tonsillar and CLL-B cells cultured with medium with or without supplementation of IL-2 or IL-4 was low (data not shown). When cultured in the CD40 system, a consistent secretion of IL-6 and IL-8 was only observed in tonsillar B cells, but not in CLL-B cells. In contrast, both tonsillar and CLL-B cells released detectable amounts of TNFa and sCD23 into the supernatants. In CLL-B cells, the addition of TGF-B increased TNF-a secretion from a mean value of 30.87 pg/ml to 96.63 pg/ml, but had no significant influence on sCD23 levels (mean values 42.94 D/ml before, and 55.13 D/ml after addition of TGF-B). In contrast, in tonsillar B cells no impact of addition of TGF-B on TNF-a secretion (mean values 160.68 pg/ml before, and 128.24 pg/ml after addition of TGF-B) was observed, whereas sCD23 levels decreased from a mean of 275.97 D/ml to 88.48 D/ml (Fig. 4). Effect of TGF-~ on costimulatory activity of CD4+ T cells

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Figure 4. Change in release of TNF-a or sCD23 into culture supernatants of tonsillar (A) and CLL-B cells (B). The grey boxes denote the mean changes in cytokine levels. Cells were cultured for 48 hours in the CD40 system supplemented with IL-2 as described in Materials and Methods. Results from 4 (3) tonsils and 5 B-CLL patients are shown.

Autocrine TGF-~ in B-CLL . 135

vation with tonsillar or CLL-B cells. Mean cytokine levels released by CD4+ T cells were higher upon coincubation with normal tonsillar B cell, than upon coincubation with CLL-B cells. Neither neutralization of endogeneous TGF-~, nor addition of exogenous TGF-~ had a significant effect on detectable amounts of IL-2 and IL-4 as secreted by irradiated and anti-CD3 activated CD4+ T cells cocultured with tonsillar or CLL-B cells in this system (Fig. 5). The addition of CLL-B cells downregulated expression of CD40L by activated CD4+ T cells. However, addition of exogeneous TGF-~ alone could not downregulate expression of CD40L, CD28, CD27, mTNF-a or CD54 by T helper cells stimulated with VIT3 or PHA (data not shown). Similar results have been reported for CD58, CD69 and CD80 (26).

Discussion In vivo, CLL-B cells are characterized by a low proliferative index and impaired apoptotic pathways. Autocrine secretion of antiproliferative cytokines such as TGF-~ could contribute to this behavior. Following this hypothesis, the in vitro effects of TGF-~ on CLL-B cells were studied under various conditions: When CLL-B cells were cultured alone or with supplementation of IL-2 or IL-4, they

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Figure 5. Release of IL-2 (A) and IL-4 (B) into culture supernatants by sublethally irradiated, anti-CD3 activated CD4+ T cells cultured for 48 hours in direct contact with tonsillar (dark bars) or CLL-B cells (light bars) as described in Materials and Methods. Mean values from 4 tonsils and 3 B-CLL patients are given. There were no significant differences between the medium control and values obtained by addition or neutralization of TGF-~ as calculated by the Friedman test.

136 . M.

SCHULER

et al.

released only small amounts of bioactive TGF-~, and neutralization of autocrine significantly enhanced B cell proliferation. Similar results were reported in CLL-B cells of some patients stimulated with IL-2 plus IL-I0, SAC, anti-p and/or mitogen plus SAC (19-21). However, most studies were hampered by a heterogenous proliferative response of CLL-B cells obtained from different patients. This heterogeneity has been attributed to variations in expression of the TGF-~ receptors (21) or downstream mediators of TGF-~ signaling (20). While culturing CLL-B cells in the CD40 system a marked proliferative response could be obtained in most cases (Fig. 3B). Surprisingly, in this setting neither autocrine nor exogenously added TGF-~ significantly influenced proliferation of clonal B cells. A similar observation has been reported in germinal center B cells stimulated via the CD40 receptor (27). This may result from the continous stimulation of the CD40 pathway by the anti-CD40 antibody presented by Ltk-cells, a condition which does not reflect the in vivo situation, where a downregulation of CD40L expression on T cells is observed approximately after 20 hours (28, 29). Whereas expression of TGF-~l RNA was not increased under stimulation in the CD40 system, a higher release of bioactive TGF-~ protein was observed (Fig. 1). From the present data, it cannot be depicted, whether this results from enhanced activation of endogenously synthesized TGF-~. However, an alternative explanation might be the increased survival of B cells cultured in the CD40 system, possibly leading to a higher amount of detectable TGF-~ in culture supernatants without altering the synthesis or activation rate per cell. When CLL-B cells were cultured in direct contact with activated CD4+ T cells, the highest basal proliferation was observed. Under these conditions, neutralization of autocrine TGF-~ had a major impact on B cell proliferation (Fig. 3C). As direct cellular contact is a prerequisite for effective stimulation of normal and malignant B cells by activated CD4+ T cells (8, 9, 30, 31), TGF-~ might modulate the surface expression of CD40L and further costimulatory molecules on activated T helper cells. Yet, in our study no significant downregulation of CD40L, CD28, CD27, CDS4 or mTNF by TGF-~ could be detected in CD4+ T cells as activated with anti-CD3 or PHA. This is consistent with observations from other groups (26). However, blocking the CD40-CD40L interaction by addition of antibodies against CD40 or CD40L resulted in a decreased proliferation of B cells cocultured with activated CD4+ T cells (10). In the B-T cell coculture system, antigenindependent costimulatory signals, as provided through CD80/CD86CD28/CTLA-4 interactions and also through CD40-CD40L interactions, are mainly responsible for the activation of CD4+ T cells (32, 33). The differences observed between normal and clonal B cells as stimulators of T cell cytokine secretion (Fig. 5) are in line with recent findings demonstrating an acquired functional anergy of T cells in CLL (34). How can these observations be transferred into the in vivo situation? Malignant B cells might be poor costimulators of CD4+ T cells, which in turn do provide improper proliferative and differentiating signals to the B cells. In concert with the deregulation of apoptotic pathways, as observed in CLL, this may lead

TGF-~

Autocrine TGF-~ in B-CLL . 137

to the characteristic accumulation of resting B cells. In addition to receptormediated down-modulation of CD40L, the vast excess of TGF-~ secreting clonal B cells could also contribute to immune dysfunction and to suppression of normal hematopoiesis in CLL (35). Both, a compromised immune system, and hematopoietic insufficiency are the mainstays of morbidity and mortality in CLL patients (1, 2). In conclusion, by using culture systems incorporating direct cellular interactions, we could define a possible role of TGF-~, as secreted by clonal B cells, in the pathophysiology of CLL. Acknowledgements We thank MARION ESSWEIN, KERSTIN KUNZ and Dr. HEIKO VAN DER KUIP for their support in the laboratory. Dr. HEINOLD GAMM is acknowledged for referring blood samples from his patients. Supported by a grant from the Deutsche Forschungsgemeinschaft (Teilprojekt A3, Sonderforschungsbereich 519).

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