Leukemia (1998) 12, 610–618 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu Malignant plasma cell lines express a functional CD28 molecule X-G Zhang1,2, D Olive3, J Devos1, C Rebouissou1, M Ghiotto-Ragueneau3, M Ferlin1 and B Klein1,2 ˆ 1Institute of Molecular Genetics, Montpellier; 2Unit for Cellular Therapy, Hopital Saint Eloi, Montpellier; and 3INSERM U 119, Marseille, France The function of CD28 molecules that are present on malignant Materials and methods plasma cells of human myeloma cell lines (HMCL) was studied. First, myeloma cells expressed a similar density of CD28 anti- gen to that of normal T cells. The myeloma CD28 molecules Antibodies and reagents were able to bind B7-Ig molecules as well as L cells transfected with a B7-1 cDNA, and anti-CD28 mAb inhibited the binding. The agonist (CD28.2) and antagonist (CD28.6) anti-CD28 Myeloma cells did not express B7-1 antigens but a low density mAbs were obtained as described elsewhere.12 MAbs against of B7-2 antigens. The myeloma B7-2 molecules of two HMCL CD3, CD16, CD25, CD56, CD69, B7-1, HLA-DR antigens, were able to bind CTLA-4 protein. No autocrine CD28:B7-2 acti- FITC-conjugated (FabЈ) fragments of goat antibodies to mouse vation could be evidenced as we found no spontaneous bind- 2 ing of the p85 subunit of PI-3 kinase to CD28 molecules. In IgG and FITC-conjugated antibodies to Fc fragments of human addition, a blocking anti-CD28 mAb did not affect the IL-6- Ig, IgG1 and IgG2 murine control antibodies (recognizing no dependent or autonomous proliferation of the HMCL. The acti- human antigens) were purchased from Immunotech vation of myeloma CD28 molecules with or without TPA stimu- (Marseille, France), mAbs to CD40, CD40L, B7-2 antigens lation did not affect the proliferation, survival, differentiation, from Pharmingen (San Diego, CA, USA), magnetic beads expression of activation antigens and cytokine receptors or coated with anti-CD19, anti-CD14 or anti-murine Ig anti- cytokine production of myeloma cells. However, the triggering of myeloma CD28 molecules by B7-1 transfectant cells resulted bodies from Dynal (Oslo, Norway). in binding of the p85 subunit of PI-3 kinase to CD28 molecules The human B7-1-human Ig and human CTLA-4-human Ig as previously shown for T cell CD28 molecules. This fusion proteins (B7-1-Ig and CTLA-4-Ig) were kindly provided expression of a large density of CD28 molecules able to bind by Dr Linsley (Bristol Myers Squibb, Seattle, WA, USA). Pur- B7 molecules might contribute to a downregulation of the ified IL-6 was provided by Dr Ytier (Ares Serono, Geneva, immune control of myeloma cells. Switzerland) and neutralizing AH-65 anti-IL-6 mAb by Dr Keywords: CD28; CD80; plasma cell; multiple myeloma Brailly (Immunotech). The B-T2 anti-gp130 mAbs were obtained by our group.13,14 PHA and purified human Ig were purchased from Sigma (St Louis, MO, USA). Rabbit antibodies Introduction to p85 subunit of PI-3 kinase or phosphotyrosine were pur- chased from UBI (Lake Placid, NY, USA) or Transduction Lab- In the process of T cell activation by antigen-presenting cell oratories (Lexington, KY, USA). Peroxidase-conjugated anti- (APC), activation of CD28 molecules present on T cells has bodies to mouse or rabbit antibodies and the ECL detection 1,2 been shown to be essential. As recently reviewed, T cell kit were purchased from Amersham (Buckinghamshire, UK). activation by APC requires at least three steps: (1) binding of Tritiated thymidine was purchased from CEA (Saclay, France). APC to T cells through adhesion molecules such as ICAM- 1:LFA-1 and LFA-3:CD2; (2) presentation of peptide antigen bound to molecules of the major histocompatibility complex Cell lines of APC to the T cell receptor; and (3) activation of the T cell CD28 molecules or related receptors by their counter recep- The XG-1 and XG-6 IL-6-dependent HMCL were obtained in tors B7-1 (CD80) or B7-2 (CD86) present on APC. A disruption our laboratory10 and the RPMI8226 and U266 HMCL were of the CD28:B7-1 interaction may result in induction of T cell purchased from ATCC (Rockville, MD, USA). The XG cells 1 anergy. CD28 activation has been shown to be critical in were cultured in culture medium (RPMI 1640 medium sup- 1,2 inducing IL-2 secretion in activated T cells. More recently, plemented with 10% fetal calf serum (FCS), 2 mML-glutamine − activation of CD28 molecules was shown to induce activation and 5 × 10 5 M 2-mercaptoethanol) and with 1 ng/ml of IL-6. 3–7 of phosphatidyl-inositol 3-kinase (PI-3) in T cells and to pro- The RPMI8226 and U266 cells were routinely cultured in cul- 8 mote survival by upregulating bcl-XL expression. ture medium without IL-6. Murine L cell fibroblasts transfected In an early report, CD28 molecules were found to be with human B7-1 cDNA (B7-1 L cell) and parental L cells 9 present on human malignant plasma cells. A high expression were generously provided by Dr A Truneh (Smith Kline of CD28 molecules on a large panel of human myeloma cell Beecham, Philadelphia, PA, USA). Human CD28 transfectant 10 lines (HMCL) or on freshly explanted myeloma cells was (DWT6.11) was obtained by transfecting full-length human 11 recently confirmed. The present study demonstrates for the CD28 cDNA in pH␤-pr-neo vector into murine T cell first time that the CD28 molecule expressed on malignant hybridoma.6 These cell lines were cultured in culture plasma cells is functional. It is able to bind its counter recep- medium. All cell lines were free of mycoplasma contami- tors, the B7 molecules, and this binding induces activation of nation as assayed using an ELISA kit from Boehringer PI-3 kinase in myeloma cells. (Mannheim, Germany). Analysis of survival of myeloma cells Correspondence: B Klein, INSERM U475, 99 Rue Puech Ville, 34100 Montpellier cedex, France; Fax 33 4 67 04 18 62 To investigate the effects of CD-28 molecule on the survival Received 7 March 1997; accepted 12 August 1997 of the human myeloma cells, XG-6 cells were washed once Functional CD28 on human plasma cells X-G Zhang et al 611 with culture medium, incubated for 5 h at 37°C in culture allogenic MLR was performed using irradiated (3000 rads) medium alone, and washed again twice to remove recombi- XG-6 HMCL cocultured at 5 × 104 cells/well with 105 allo- nant bound IL-6. Cells (3 × 105 cells/ml) were then cultured genic T cells in 96-well round-bottomed plates in RPMI1640 with either culture medium alone or with the activating and 5% heat-inactivated human AB serum. When indicated, CD28.2 anti-CD28 mAb (10 ␮g/ml) or with 1 ng/ml of IL-6. A agonist (CD28.2) or antagonist (CD28.6) anti-CD28 mAbs or mouse IgG1 anti-CD3 mAb was used as a control antibody. their isotypic controls were added at 10 ␮g/ml. Purified T cells After 24, 48 and 72 h of culturing, the percentage of viable were also cultured with PHA (1 ␮g/ml). After 5 days of cocul- cells was determined by the trypan blue dye exclusion ture, cells were pulsed with 1 ␮Ci of tritiated thymidine for method, and apoptotic cells were detected by propidium 12 h, harvested and counted. All microculture tests were car- iodide staining and flow cytometry. Apoptotic cells had a ried out in triplicate. DNA content inferior to that of cells in the G1 phase of the cell cycle. Proliferation and immunoglobulin production of myeloma cells Flow cytometry To investigate the effect of activation of CD28 molecules on Indirect immunofluorescence was done by incubating 5 × 105 the proliferation of myeloma cells, cells were washed once cells with 1 ␮g of murine mAb or soluble B7-1-Ig or soluble with culture medium, incubated for 5 h at 37°C in culture CTLA-4-Ig in 100 ␮l of staining buffer consisting of PBS with medium alone to remove bound IL-6, and washed again 0.25% bovine serum albumin and 0.01% sodium azide for 45 twice. Myeloma cells were then cultured with various concen- min at 4°C. Isotype-matched Ig was used as a control (human trations of the CD28.2 activating anti-CD28 mAb or control or murine IgG). Cells were then stained with either FITC-con- mAb with or without TPA (100 ng/ml; Sigma) in 96-well flat- Ј jugated (Fab )2 fragments of goat antibodies to mouse IgG or bottomed microplates for 2 or 5 days. We used a mouse IgG1 FITC-conjugated antibodies to Fc fragments of human Ig. Flow anti-CD3 mAb that did not bind to myeloma cells as a control cytometry was performed with a FACScan apparatus (Becton antibody. In some experiments, the effect of the blockage of Dickinson, Palo Alto, CA, USA). Cells were considered as CD28 activation by the CD28.6 neutralizing anti-CD28 mAb positive when the fluorescence intensity of the labelling was was investigated by adding 10 ␮g/ml of the anti-CD28 mAb greater than the mean fluorescence intensity + 2s.d. obtained in 5-day cultures of XG cells with IL-6 (100 pg/ml) or of with a control mAb. RPMI8226 and U266 cells. At the end of the culture, 0.5 ␮Ci of tritiated thymidine (25 Ci/mM; CEA, Saclay) were added to each culture well for 8 h and tritiated thymidine incorporation CD28 adhesion assays was determined on six replicate culture wells.