CD40 Triggering of Chronic Lymphocytic Leukemia B Cells Results

CD40 triggering of chronic lymphocytic leukemia B cells results in efﬁcient alloantigen presentation and cytotoxic T lymphocyte induction by up-regulation of CD80 and CD86 costimulatory molecules LE Van den Hove1, SW Van Gool1,2, P Vandenberghe3, M Bakkus4, K Thielemans4, MA Boogaerts3 and JL Ceuppens1

1Laboratory of Experimental Immunology, 2Division of Pediatrics, and 3Division of Hematology, University Hospital Gasthuisberg, Catholic University of Leuven, Leuven; and 4Laboratory of Physiology, Free University of Brussels, Brussels, Belgium

Freshly collected chronic lymphocytic leukemia B cells (B-CLL APC.11 Moreover, transfection of B7-1 (CD80) or B7-2 (CD86) cells) are known to be inefficient at stimulating allogeneic T into tumor cells strongly enhances antitumor T cell responses cells, and to lack significant expression of B7 (CD80 and CD86) in murine models.12–15 Considering that normal resting B cells costimulatory molecules. We investigated the potential of CD40 16 triggering to up-regulate the expression of adhesion and acquire antigen-presenting capacity only after activation, costimulatory molecules on B-CLL cells, and to enhance their that B-CLL cells morphologically resemble small resting B immunogenicity towards allogeneic T cells. B-CLL cells cocul- cells, and that they lack significant surface expression of B7 tured with human CD40 ligand-expressing mouse fibroblasts costimulatory molecules,9,17 T cell unresponsiveness towards rapidly up-regulated CD54 and CD58 adhesion molecules and autologous B-CLL cells may at least partially be explained by B7-1 (CD80) and B7-2 (CD86) costimulatory molecules, and acquired a strong stimulatory capacity towards CD4+ as well an inadequate costimulatory capacity of this tumor. We there- as isolated CD8+ allogeneic T cells. Costimulation by both CD80 fore intended to enhance the immunogenicity of B-CLL cells and CD86 proved critical for allogeneic T cell proliferation and by up-regulating B7 expression, making use of a physiological CD25 and HLA-DR expression, since these were strongly control mechanism: CD40–CD40 ligand (CD40L) interaction. inhibited by anti-CD80 or anti-CD86 monoclonal antibodies, and CD40 is a receptor belonging to the tumor necrosis factor completely abrogated by CTLA4-Ig fusion protein, which blocks (TNF)-receptor superfamily. Interaction of CD40 on B cells both CD80 and CD86. B7 costimulation also proved critical for with its ligand CD40L on activated T cells was found to play restimulation of primed B-CLL-reactive T cells. Most 18 importantly, priming of purified CD8+ T cells with CD40-trig- a key role in B cell activation, survival, and differentiation. gered allogeneic B-CLL cells resulted in cytotoxic activity Both normal and malignant B cells can be activated via CD40 against the unstimulated B-CLL cells. These findings raise the to upregulate B7 expression19–23 and to acquire immuno- possibility that CD40 triggering of B-CLL cells might be genicity towards allogeneic T cells.20,22,23 We therefore inves- exploited in immunotherapeutic protocols. tigated whether triggering of B-CLL cells with CD40L-trans- Keywords: B-CLL; CD40; CD40L; CD80; CD86; costimulation fected cells induces them to become efficient APC for the generation of allogeneic proliferative and cytotoxic T cell responses, and whether these alloantigen-primed T cells are Introduction capable of exerting cytotoxic activity against unstimulated B- CLL cells. Our findings indicate that CD40 should be Chronic lymphocytic leukemia of B cell origin (B-CLL) is the considered as a potential target for immunotherapy. most common leukemia in the western hemisphere, account- ing for almost one third of all cases.1 The disease is incurable with conventional treatment programs, and patients with Materials and methods advanced B-CLL have a mean survival of less than 5 years.2 New approaches, including treatment with cytokines such as Patients interferon-␣ (IFN-␣), have not been very effective.3,4 The recent demonstration of major histocompatibility com- Peripheral blood samples were collected from five healthy plex (MHC)-restricted recognition of autologous B-CLL cells donors and from five newly diagnosed patients with B-CLL. 5,6 by tumor-specific T cells indicates that B-CLL cells poten- B-CLL was diagnosed on the basis of clinical, morphological, tially present tumor-specific peptides (mutant proteins, and immunophenotypic features. idiotypic determinants of the tumor immunoglobulin) in the context of MHC molecules. However, only sparse evidence exists that tumor cells in general can induce a clinically sig- Monoclonal antibodies and other reagents nificant antitumor T cell response in the autologous host.7 Moreover, B-CLL cells are inefficient stimulators even for allo- 8,9 The mouse monoclonal antibodies (mAb) used for flow cyto- geneic T cells. Besides the interaction between peptide metry were anti-CD3, anti-CD4, anti-CD8, anti-CD11a (LFA- antigen/MHC on antigen-presenting cells (APC) and the anti- 1), anti-CD19, anti-CD25, anti-CD54 (ICAM-1), anti-CD80 gen-specific T cell receptor, T cells are known to require at 10 (anti-B7-1), anti-kappa, anti-lambda, anti-HLA-DR (MHC class least one accessory signal for activation. This second signal II), and the appropriate mouse immunoglobulin (Ig) isotype is delivered by costimulatory molecules on the APC through 10 controls, from Becton Dickinson (Erembodegem, Belgium). their counter-receptors on the T cells. Particular interest has Anti-CD50 (ICAM-3) was from Biosource International recently been shown in the members of the B7 family, which (Camarillo, CA, USA). Anti-CD58 (LFA-3) was from Immuno- appear to be the most important costimulatory molecules on tech (Marseille, France). The anti-B7-2 mAb FUN-1 (anti- CD86) was from Pharmingen (San Diego, CA, USA). Anti- Correspondence: J Ceuppens, Laboratory of Experimental Immu- MHC class I was from One Lambda (Canoga Park, CA, USA). nology, Campus Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium The hybridoma producing mAb OKT3 (anti-CD3) used for Received 23 October 1996; accepted 11 December 1996 anti-CD3 redirected cytotoxicity assays, was obtained from CD40 triggering of chronic lymphocytic leukemia B cells LE Van den Hove et al 573 the ATCC (Rockville, MD, USA). The anti-CD80 (clone B7- and 3T6 cells transfected with human type II Fc␥R (CD32)24 24) mAb used in blocking experiments, was a gift from Immu- were a kind gift from Dr J van de Winkel (University of nogenetics (Gent, Belgium). The anti-CD86 (clone IT2.2) mAb Utrecht, Utrecht, The Netherlands). used in blocking experiments, was purchased from Pharm- The 3T6/CD40L cell line was obtained by transfection of ingen. CTLA4-Ig fusion protein was a gift from Dr A Lanzavec- 3T6 cells with the BCMGSneo-CD40L plasmid (kindly pro- chia (Basel Institute for Immunology, Basel, Switzerland). vided by Dr D Graf, Max-Planck Institute, Erlangen, Germany) containing the cDNA for human CD40L,25 by lipofection (DOTAP; Boehringer Mannheim). The transfected cells were Immunofluorescence studies selected in the presence of 200 ␮g/ml G418-sulphate (Gibco). The day before transfection, cells were seeded in a 10 cm dish Three- and two-color analyses were performed by direct at 8 × 105 cells per dish. Transfection was performed with immunofluorescence with FITC (fluorescein isothiocyanate)-, 7.5 ␮g plasmid. Medium was replaced after 16 h. Selection PE (phycoerythrin)-, and PerCP (peridinin chlorophyll medium was added after 24 h. Neo-resistant clones were protein)-conjugated mAb. Briefly, 100 ␮l cell suspension selected and screened for CD40L expression by staining with (cultured lymphocytes or peripheral blood, eventually diluted affinity-purified CD40-IgM (3 ␮g/ml; the CD40-IgM-produc- with phosphate-buffered saline to a maximum of 10 × 106 ing hybridoma was kindly provided by Dr P Lane, Basel cells/ml) was incubated with the directly conjugated mAb. Institute for Immunology, Basel, Switzerland), followed by a After 15 min, the cells were either washed (cultured biotinylated anti-hIgM mAb (1D12, IgG1) or an irrelevant lymphocytes), or incubated with FACS lysing solution (Becton mouse IgG1 mAb (3G5, IgG1) and streptavidin-PE (Becton Dickinson) for 10 minutes to lyse the erythrocytes (peripheral Dickinson). The cells were then analysed on a FACStar blood). The cells were then centrifuged and fixed in 0.5 ml of instrument (Becton Dickinson). a 0.5% paraformaldehyde/PBS solution. Two- and three-color immunofluorescence analyses were performed on a FACScan flow cytometer (Becton Dickinson). Data obtained by flow Activation of B-CLL cells by coculture with cytometry were analyzed by gating the lymphocytes using for- 3T6/CD40L mouse fibroblasts ward and side light scatter in the Lysis II software program (Becton Dickinson). Ten thousand cells were routinely PBMC containing 1 × 106 B-CLL cells were cultured for 22 h analyzed for each marker combination. in complete medium, with 0.375 × 106 mitomycin C (50 ␮g/ml)-treated 3T6/CD32 mouse fibroblasts, or 0.375 × 106 mitomycin C-treated 3T6/CD40L mouse fibro- Cell separation blasts in 24-well flat-bottom Falcon tissue culture plates (Becton Dickinson) in 1 ml of medium. All cultures were per- ° Peripheral blood mononuclear cells (PBMC) were isolated by formed at 37 C in a 5% CO2 atmosphere. After coculture, the centrifuging the blood over a Lymphoprep (density 1.077) cells were collected and purified by magnetic immunoselec- gradient (Nycomed, Oslo, Norway). The cells were resus- tion using anti-CD19-coated Dynabeads (Dynal) and pended in RPMI 1640 culture medium (Gibco), supplemented Detachabead (Dynal) for immunophenotyping and coculture with 2 mML-glutamine (Gibco); 100 U/ml penicillin (Gibco); with allogeneic T cells. 100 ␮g/ml streptomycin (Gibco); and 10% iron-supplemented bovine calf serum (BCS; Hyclone, Logan, UT, USA). For purification of CD3+ lymphocytes, monocytes were first removed Stimulation of allogeneic T cells with B-CLL cells by cold agglutination, by rotating the tubes for 30 min at 4°C. The agglutinated monocytes were sedimented out and the For primary stimulation, purified CD4+, CD8+, or CD3+ T cells lymphocytes were further purified using complement-fixing were cultured with irradiated (40 Gy) B-CLL cells at a 1/4 anti-NK, anti-monocyte mAb and Lympho-KWIK-T (One stimulator/responder ratio for 5–7 days. For inhibition experi- Lambda, Los Angeles, CA, USA). For purification of CD4+, ments, anti-CD80 or anti-CD86 mAb (both at 2 ␮g/ml, final CD8+ and CD19+ lymphocytes, magnetic immunoselection on concentration), or CTLA4-Ig fusion protein (10 ␮g/ml, final PBMC with anti-CD4-, anti-CD8-, and anti-CD19-coated concentration) were added from the beginning of the culture Dynabeads (Dynal, Oslo, Norway) respectively, was used. period. After 5–7 days, the cells were harvested and resus- The beads were applied with a bead-to-target ratio of 3:1 for pended in medium for immunophenotyping and analysis of CD4+ and CD8+ lymphocytes, and a bead-to-target ratio of cytotoxic capacity, or for restimulation. In the latter case, the 5:1 for CD19+ lymphocytes. The selected cells were detached cells were first rested in medium during 1 day in the absence from the beads using Detachabead solution (Dynal). Greater of the stimulator cells. The T cells were then restimulated with than 95% purification was achieved for CD4+ and CD8+ cells, irradiated tumor cells at a 1/4 stimulator/responder ratio. and greater than 99% purification was achieved for CD3+ and Three to four days later, the cells were harvested and resus- CD19+ cells. The purified CD19+ B cells of our patients pended in medium for immunophenotyping and analysis of consisted nearly entirely (Ͼ95%) of tumor cells, as evidenced cytotoxic capacity. by anti-kappa/anti-lambda/anti-CD19 three-color immuno- To measure the effect of allogeneic stimulation with B-CLL fluorescence. cells on T cell proliferation, 0.25 × 106 T cells were cultured with 0.0625 × 106 B-CLL cells in 96-well flat-bottomed Falcon tissue culture plates (Becton Dickinson) in 200 ␮l of culture Cell lines medium per well. During the last 8 h of culture (on days 5–7 for primary stimulations, on days 3–4 for restimulations), the The P815 cell line (obtained from the ATCC) is an NK-resistant cells were pulsed with 1 ␮Ci 3H-thymidine (Amersham Inter- DBA/2-derived murine mastocytoma cell line that expresses national, Amersham, UK). Proliferation of T cells was evalu- mouse Fc␥RII and Fc␥RIII. The mouse fibroblast 3T6 cell line ated by measuring 3H-thymidine incorporation and was CD40 triggering of chronic lymphocytic leukemia B cells LE Van den Hove et al 574 expressed as the mean counts per minute (c.p.m.) of Table 1 Proliferative responsesa of allogeneic T cells after culture quadruplicate wells. with irradiated unstimulated B-CLL cells or CD40-triggered B-CLL cells

Cytotoxicity assays B-CLL Responder Stimulator cellsc from T cellsb patient Noned Unstimulated CD40-triggered Cytotoxic activity was measured in a 4-h target cell lysis assay number using murine P815 cells or unstimulated B-CLL cells as targets. B-CLL B-CLL One million targets were incubated with 200 ␮Ci of 51Cr + e ± ± sodium chromate (Amersham International, Amersham, 1 CD8 ND 1097 486 21 863 5581 2 CD8+ 162 ± 19 212 ± 168 1275 ± 445 England) for 1–2 h in the case of P815 target cells and over- CD4+ 172 ± 77 397 ± 261 24 607 ± 3582 night in the case of B-CLL cells. The cytolytic assays were 3 CD8+ 294 ± 143 198 ± 79 1820 ± 867 performed in a 96-well, V-bottomed microtiter plate at an CD4+ 250 ± 244 259 ± 102 6074 ± 1670 effector:target ratio of 20:1. Five thousand 51Cr-labeled target 4 CD3+ 151 ± 40 476 ± 479 43 068 ± 5646 cells were mixed with 105 effectors in a total volume of 200 ␮l medium. Four wells were filled with 5 × 103 targets in 200 ␮l aProliferation was measured as 3H-thymidine incorporation during alone, and four wells with 5 × 103 targets in 100 ␮l medium the last 8 h of culture on days 5–7 of culture and expressed as plus 100 ␮l saponin (for evaluation of spontaneous and maxi- c.p.m. bResponder T cells (from a different donor in each experiment) mal release, respectively). Since P815 cells are NK-resistant, were purified with Dynabeads (CD4+ and CD8+ T cells) or Lympho- their lysis reflects ‘lymphokine-activated killer (LAK)-like’ KWIK-T (CD3+ T cells) before initiation of the cultures. activity of cytotoxic T cells.26,27 The microtiter plates were cStimulator B-CLL cells were purified with Dynabeads. centrifuged for 10 min at 200 g and incubated for 4 h at 37°C dAs a control, culture of allogeneic T cells in medium was used. ␮ eND, not determined. in a CO2 incubator. Afterwards, 100 l of the supernatant of each well was collected and counted in a gamma counter. 51 Results are expressed either as percentage Cr release of P815 CD50, CD54 and CD58 adhesion molecules, and of CD80 51 cells (for evaluation of the LAK activity), or as percentage Cr and CD86 costimulatory molecules on peripheral blood B- release of unstimulated B-CLL cells (for evaluation of the CLL cells. As shown in Table 2, peripheral blood B-CLL cells tumor-directed cytotoxicity). The following formula was used nearly completely lacked expression of the CD11a adhesion to calculate the cytotoxic activity: molecule, but strongly expressed the CD50 adhesion mol- experimental release − spontaneous release ecule and MHC class I and class II molecules. Most tumor ͩ ͪ × 100. cells of four of the five patients also dimly expressed the CD54 maximal − spontaneous release and CD58 adhesion molecules and the CD80 and/or CD86 costimulatory molecules. The tumor cells of one patient Results (patient 3) were largely CD54 negative, and nearly completely lacked expression of CD80 and CD86 costimulatory mol- CD40-triggered B-CLL cells, unlike resting B-CLL cells, ecules. As shown in Figures 2 and 3, the CD40-triggered B- induce significant proliferative responses in purified CLL cells significantly up-regulated expression levels of CD80 CD4+ as well as CD8+ allogeneic T cells and CD86 costimulatory molecules (the results for the five patients are depicted in Figure 2, bold solid line), and of CD54 PBMC were isolated from the blood of four newly diagnosed and CD58 adhesion molecules (Figure 3). The expression of B-CLL patients, and cultured overnight in medium or with CD50 was only marginally higher, and the expression of CD40L-transfected mouse fibroblasts. Unstimulated B-CLL CD11a remained low. In contrast, B-CLL cells cocultured with cells and CD40-stimulated B-CLL cells were immunomagnet- CD32-transfected 3T6 cells (control stimulators) displayed a ically purified, irradiated (40 Gy) and used as stimulators for slightly higher expression of CD54 than unstimulated B-CLL purified CD4+, CD8+, or CD3+ allogeneic T cells from healthy cells, while the other adhesion and costimulatory molecules donors. As shown in Table 1, only the CD40-triggered B-CLL were unchanged (Figure 3). This indicates that CD40L mol- cells were capable of inducing allogeneic T cell proliferation. ecules were specifically responsible for the strong upregul- Interestingly, both purified CD4+ and (to a lesser extent) CD8+ ation of CD54, CD58, CD80 and CD86 and CD40-triggered allogeneic T cells demonstrated significant proliferation upon B-CLL cells. stimulation with CD40-triggered B-CLL cells. In agreement with this, CD25 and HLA-DR activation marker expression on both CD4+ and CD8+ allogeneic T cells was significantly B7 costimulatory molecules are necessary for the increased after culture of the respective subsets with CD40- induction of allogeneic T cell proliferative responses triggered but not with resting B-CLL cells (Figure 1a and b). towards B-CLL cells Despite their low proliferation, the upregulation of CD25 and HLA-DR expression clearly shows that isolated CD8+ T cells The two signal theory of T cell activation predicts that the were efficiently stimulated by CD40-triggered B-CLL cells. expression of costimulatory molecules limits the generation of T cell responses.28 We therefore studied the role of B7 costimulatory molecules during the induction of allogeneic T Cell surface expression of adhesion and costimulatory cell activation by CD40-triggered B-CLL cells. Inhibition molecules on peripheral blood B-CLL cells and on experiments were performed by adding control mAb, anti- CD40-triggered B-CLL cells CD80 mAb, anti-CD86 mAb or CTLA4-Ig fusion protein at the initiation of the primary culture. Figure 4a shows that CTLA4- In view of the known importance of costimulatory signals for Ig fusion protein nearly completely blocked the proliferative T cell activation, we next studied the expression of CD11a, response of allogeneic T cells towards CD40-triggered B-CLL CD40 triggering of chronic lymphocytic leukemia B cells LE Van den Hove et al 575 Table 2 Expression of adhesion, costimulatory, and MHC molecules on peripheral blood B-CLL cellsa

Surface Mean ﬂuorescence intensity of B-CLL Mean ± molecule cellsb (Percentage of B-CLL cells) s.d.

Patient Patient Patient Patient Patient 12345

CD11a 5 4 9 6 6 6 ± 2 (4) (0.5) (5) (4) (7) (4 ± 2) CD50 337 323 304 378 333 335 ± 27 (100) (100) (100) (100) (100) (100) CD54 35 76 12 43 41 41 ± 23 (79) (100) (22) (97) (96) (79 ± 33) CD58 84 34 43 28 92 56 ± 30 (100) (85) (87) (59) (99) (86 ± 17) CD80 3 16 3 25 7 11 ± 10 (8) (53) (0.5) (76) (16) (31 ± 32) CD86 46 17 7 48 82 40 ± 29 (81) (70) (3) (47) (91) (58 ± 35) MHC 520 559 513 1082 916 718 ± 264 class I (100) (100) (100) (100) (100) (100) MHC 1649 960 1794 2499 1857 1752 ± 550 class II (100) (100) (100) (100) (100) (100)

aAs determined by direct three-color immunoﬂuorescence on whole blood. bMean ﬂuorescence intensity of mouse IgG controls was between 4 and 15.

demonstrate that B7 costimulatory molecules are also crucial for restimulation of primed B-CLL-reactive T cells, and that Figure 1 Expression of CD25 and HLA-DR activation markers on CD4+ (a) and CD8+ (b) allogeneic T cells after culture of the respective the low expression of B7 costimulatory molecules on unstimu- purified T cell subsets with unstimulated (unst B-CLL) or CD40-trig- lated B-CLL cells is still insufficient to drive the expansion of gered (CD40-tr B-CLL) B-CLL cells. As a control, CD4+ and CD8+ allo- primed B-CLL-reactive T cells. geneic T cells were cultured in medium alone. The CD4+ and CD8+ T cells from normal donors were purified with Dynabeads before allogeneic stimulation. CD25 and HLA-DR expression was measured by three-color immunofluorescence FACS analysis. Results are expressed + as fluorescence intensities in arbitrary units (mean ± s.d.) of experi- CD40-triggered B-CLL cells induce allogeneic CD8 T ments with T cells from normal donors, and tumor cells of patients 2 cell cytotoxicity against unstimulated B-CLL cells and 3 (a) and patients 1, 2 and 3 (b), respectively. We next studied whether CD40-triggered B-CLL cells have the cells (99%, 91% and 99% inhibition, respectively, in three capacity to generate allogeneic T cell cytotoxicity. Isolated experiments performed). Anti-CD80 mAb alone inhibited the CD4+ and CD8+ T cells were cultured with either unstimulated proliferative T cell response towards the stimulated tumor or CD40-triggered B-CLL cells for 5–7 days. Cytotoxic activity cells by 81%, 53% and 98%, respectively. Anti-CD86 mAb of the allogeneic T cells was investigated immediately after had a variable effect (from 0 to 88%). This variability corre- culture in two different cytotoxicity assays. LAK-like activity lates with the relatively low CD86 expression level (mean of the cytotoxic effector cells was detected by lysis of P815 fluorescence intensity 51) on the CD40-triggered B-CLL cells cells. Resting B-CLL cells were used as a target to measure of patient 3, when compared with the stimulated tumor cells cytotoxic T lymphocyte (CTL) activity generated towards the of patients 2 (mean fluorescence intensity 203) and 4 (mean tumor. As shown in Table 3, no CTL activity was generated fluorescence intensity 243). The expression levels of CD11a, when T cells were cultured with unstimulated B-CLL stimu- CD50, CD54, CD58, and CD80 molecules on the CD40-trig- lator cells. In contrast, when CD40-triggered B-CLL cells were gered tumor cells of the three patients were similar (data not used as stimulators and CD8+ T cells as responders, CTL shown). Two-color FACS analysis of the responding T cells activity was generated against the unmanipulated tumor cells further demonstrated that CTLA4-Ig strongly interfered with of the three patients investigated. On two occasions, CD40- the upregulation of CD25 and HLA-DR activation markers triggered B-CLL cells were used to generate CTL activity in when compared to the conditions with anti-CD80 mAb or both CD4+ and CD8+ allogeneic T cells. As shown in Table 3, anti-CD86 mAb alone (Figure 4b). In a separate experiment, CTL activity was only detected within the CD8+ T cell subset. the proliferative response of primed CD3+ T cells during These results show that isolated CD8+ T cells can be triggered restimulation with CD40-triggered B-CLL cells of patient 4 to develop CTL activity in the absence of help from CD4+ T (35587 ± 5530 c.p.m.) was also completely blocked by the cells, and that once activated, these CD8+ CTL can lyse their addition of CTLA4-Ig (357 ± 67 c.p.m.). These observations targets in the absence of B7–CD28 interaction. CD40 triggering of chronic lymphocytic leukemia B cells LE Van den Hove et al 576

Figure 3 Cell surface expression (mean ﬂuorescence intensity) of adhesion and costimulatory molecules on B-CLL cells cultured for 22 h in medium (unstimulated), on B-CLL cells cocultured for 22 h with CD32-transfected 3T6 cells, and on B-CLL cells cocultured for 22 h with CD40L-transfected 3T6 cells. Cell surface expression of adhesion and costimulatory molecules was determined by three-color immunoﬂuorescence FACS analysis. Results are expressed as mean ± s.d. of experiments with tumor cells from patients 2, 3 and 4.

cells proliferated upon restimulation with CD40-triggered B- CLL cells, while their response towards unstimulated B-CLL cells was low to absent. CD8+ T cells primed with CD40-triggered tumor cells of patient 1 (LAK activity = 0 ± 2%; tumor- directed cytotoxicity = 21 ± 21%) demonstrated similarly enhanced tumor-directed CTL activity upon restimulation with either the stimulated B-CLL cells (LAK activity = 3 ± 2%; tumor-directed cytotoxicity = 39 ± 26%) or with the unstimulated B-CLL cells (LAK activity = 3 ± 2%; tumor-directed cytotoxicity = 40 ± 11%). This observation suggests that once activated, boosting of CTL effector function is relatively independent of costimulatory signals.

Culture of allogeneic T cells with unstimulated B-CLL Figure 2 Cell surface expression of CD80 (left column) and CD86 cells does not cause unresponsiveness or anergy (right column) costimulatory molecules on freshly collected, unstimulated (thin solid line) and on CD40-triggered B-CLL cells (bold solid Since the unstimulated B-CLL cells demonstrate weak line) from the five patients. CD40-triggered B-CLL cells are cells cocul- expression levels of CD80 and CD86 costimulatory mol- tured with CD40L-transfected 3T6 mouse fibroblasts for 22 h. FACS ecules, such tumor cells might induce tumor-specific anergy histograms show the relative cell number vs the fluorescence intensity in vivo. To investigate this possibility, we tried to induce (on a 4 log scale). The dotted line shows the staining with control mouse IgG. anergy in allogeneic T cells in vitro, by coculturing them with irradiated unstimulated B-CLL cells. After 5–7 days, the allogeneic T cells were harvested and rested overnight in medium, B7 costimulatory molecules are crucial for and then restimulated with CD40-triggered B-CLL cells. restimulating proliferative responses of primed B-CLL- Figure 6 shows that following culture with unstimulated B-CLL reactive CD8+ T cells, but not for boosting of CTL cells, a strong allogeneic T cell proliferative response was activity detected in response to restimulation with CD40-triggered B- CLL cells. These experiments clearly demonstrate that the The previous results indicate that B7–CD28 interaction is not unstimulated B-CLL cells did not induce allospecific T cell required for the effector phase of CTL activity by CD8+ T cells. anergy during in vitro culture. To determine whether previously activated CD8+ T cells require B7 costimulatory signals for restimulation, a secondary culture was performed with unstimulated and CD40-triggered Discussion B-CLL cells. Following primary stimulation of purified CD8+ T cells with CD40-triggered B-CLL cells for 5–7 days, T cells We demonstrate in this paper that CD40 triggering of B-CLL were harvested and rested overnight in medium. The T cells cells upregulates their expression of adhesion and costimu- were recultured with irradiated stimulator cells for 3–4 days latory molecules, and strongly enhances their capacity to and T cell proliferation (n = 2) and cytotoxicity (n = 1) was induce allogeneic T cell proliferation and tumor-directed cyto- determined. Figure 5 shows that primed allogeneic CD8+ T toxicity. CD40 triggering of chronic lymphocytic leukemia B cells LE Van den Hove et al 577 Table 3 Cytotoxic activity of allogeneic T cells after culture with unstimulated B-CLL cells or with CD40-triggered B-CLL cells

Patient Responder Stimulator cellsb % killing ofc T cellsa P815d CLLe

1 CD8+ unst B-CLL 0 ± 00±4 CD8+ CD40-tr B-CLL 0 ± 221±21 2 CD4+ unst B-CLL 3 ± 25±11 CD4+ CD40-tr B-CLL 2 ± 26±2 CD8+ CD40-tr B-CLL 10 ± 153±1 3 CD4+ unst B-CLL 0 ± 30±7 CD4+ CD40-tr B-CLL 0 ± 50±8 CD8+ unst B-CLL 0 ± 00±5 CD8+ CD40-tr B-CLL 51 ± 825±12

aResponder T cell subsets were puriﬁed with Dynabeads before initiation of the culture. bStimulator B-CLL cells were puriﬁed with Dynabeads. c% Cytotoxicity after the 5–7 day primary culture was evaluated by 51Cr-release assays. The results represent lysis at an effector:target ratio of 20:1. Results are expressed as mean ± s.d. of triplicate experiments. dKilling of P815 cells is used to measure LAK activity. eKilling of unstimulated B-CLL cells is used to measure tumor- directed cytotoxicity. unst B-CLL, unstimulated B-CLL cells; CD40-tr B-CLL, CD40-triggered B-CLL cells.

Figure 4 The proliferative response and activation marker expression on allogeneic T cells during culture with CD40-triggered B-CLL cells is reduced by blocking of B7 costimulatory molecules. Inhibition experiments were performed by adding control mAb, anti- CD80 mAb, anti-CD86 mAb, or CTLA4-Ig fusion protein at the beginning of the cultures. Inhibition of allogeneic T cell proliferation (a) + and activation marker expression (b) is shown. CD4 (experiments 1 + + Figure 5 Activated CD8 T cells still require B7–CD28 interaction and 2) and CD3 T cells (experiment 3) were from normal donors for restimulation. The proliferative response of allogeneic CD8+ T cells and were purified with Dynabeads and Lympho-KWIK-T, respectively. was measured upon restimulation with either CD40-triggered (CD40- 3 Proliferation was measured as H-thymidine incorporation during the tr B-CLL) or unstimulated (unst B-CLL) B-CLL cells, after primary cul- last 8 h of culture on days 5–7 of the culture. CD25 and HLA-DR ture with CD40-triggered B-CLL cells. As a control, culture of allog- expression (mean fluorescence intensity) was measured by three-color eneic CD8+ T cells in medium was used. The CD8+ T cells from two immunofluorescence FACS analysis of the allogeneic T cells. Experi- normal donors were purified with Dynabeads. Proliferation in the sec- ments 1–3 were performed with the CD40-triggered B-CLL cells of ondary culture was measured as 3H-thymidine incorporation during patients 2, 3 and 4, respectively, as stimulators. the last 8 h of the culture. Experiments 1 and 2 were performed with the tumor cells of patients 1 and 2, respectively, as stimulators. Other investigators also observed that fresh B-CLL cells are unable to stimulate allogeneic T cell proliferation despite clear CD8+ allogeneic T cells. The latter observation confirms and expression of MHC class I and class II molecules.8,9,20 Further- extends the findings of Yellin et al20 with total CD3+ T cells, more, resting B cells from healthy donors are equally inef- and demonstrates that CD40-triggered B-CLL cells can trigger ficient stimulators for allogeneic T cells.29 The dim expression significant proliferation of CD8+ (allogeneic) T cells in the of CD54 and CD58 adhesion molecules and of CD80 and absence of CD4+ T cell help. The expression of both CD80 CD86 costimulatory molecules on both normal resting B cells and CD86 costimulatory molecules appeared important for and B-CLL cells is apparently insufficient for efficient T cell allogeneic T cell proliferation, since this was nearly com- activation. pletely inhibited by anti-CD80 and/or anti-CD86 mAb alone CD40 triggering of B-CLL cells with CD40L-expressing in two of the three experiments, and was completely abro- mouse fibroblasts resulted in a rapid upregulation of CD80 gated by CTLA4-Ig fusion protein, which blocks both CD80 and CD86 costimulatory molecules, and strongly enhanced and CD86. These observations suggest that both costimulatory their stimulatory capacity towards purified CD4+ as well as molecules contribute to reach a critical ‘costimulatory thresh- CD40 triggering of chronic lymphocytic leukemia B cells LE Van den Hove et al 578 cytotoxic activity in isolated CD4+ and CD8+ allogeneic T cell subsets, tumor-directed cytotoxicity was only induced within the CD8+ T cell subset. This is in agreement with Plumas et al,33 who recently observed that the CTL activity of allogeneic T cells against malignant B cells from non-Hodgkin’s lymphomas was exerted by CD8+ T cells. Moreover, in our in vitro experiments cytotoxicity was induced within purified CD8+ T cells, indicating that cytotoxicity generation by CD40-triggered B-CLL cells may also occur without CD4+ T cell help. Direct costimulation of CD8+ T cells by B7-positive tumor cells has been observed in vivo too.13 These findings clearly illustrate the importance of B7 expression for the induction of CTL responses. The secondary response of primed B-CLL-reactive T cells towards CD40-triggered B-CLL cells was nearly completely abolished by CTLA4-Ig, indicating that B7 costimulation is also crucial for restimulation of pre-activated B-CLL-reactive T cells. Furthermore, primed B-CLL-reactive T cells demonstrated a very low secondary proliferative response upon restimulation with resting B-CLL cells, illustrating that the dim expression of CD80 and CD86 costimulatory molecules on resting B-CLL cells is insufficient to stimulate primary as well as secondary T cell proliferative responses. This is in contrast to the B7-independent secondary responses of pre-activated T cells towards recall antigens presented by monocytes,36 which may relate to presentation of other costimulatory molecules by the monocytes. However, using CD40-triggered follicular lymphoma cells as primary stimulators, Schultze et al22 also found a decreased dependence on costimulatory molecules to subsequently drive the proliferation of primed allogeneic T cells. On the other hand, we observed that once allogeneic T cell cytotoxicity was generated by the CD40-triggered B-CLL cells, this was maintained or even enhanced after a secondary culture period with unstimulated tumor cells. This confirms

+ + the observation that CTL effector function, once activated, is Figure 6 Proliferative responses of CD8 (a) and CD3 (b) allog- 27,37 eneic T cells on restimulation with CD40-triggered B-CLL cells, after relatively independent of costimulatory signals. primary culture with either unstimulated B-CLL (unst B-CLL) cells or Until now, our attempts to induce autologous T cell acti- CD40-triggered B-CLL (CD40-tr B-CLL) cells. CD8+ and CD3+ T cells vation with CD40-triggered B-CLL cells were unsuccessful from normal donors were purified with Dynabeads and Lympho- (unpublished data). Supplementation of IL-2 and/or IL-4 to the KWIK-T, respectively, before the primary culture. Proliferation in the cultures equally proved ineffective to induce tumor-specific 3 secondary culture was measured as H-thymidine incorporation dur- cytotoxic activity of autologous T cells. One possibility is that ing the last 8 h of the culture. The experiments were performed with the tumor cells of patients 1 (a) and 4 (b) as stimulators. the unresponsiveness of autologous T cells against CD40- stimulated B-CLL cells resulted from previous in vivo induction of tumor-specific anergy by the massive amount of tumor old’ for T cell activation. Furthermore, since the dim cells usually present in CLL patients. Although we failed to expression of CD86 on the stimulated B-CLL cells of patient induce allospecific anergy in allogeneic T cells in vitro by 3 was still sufficient for allogeneic T cell activation in the pres- coculture with irradiated unstimulated B-CLL cells at a stimu- ence of anti-CD80 mAb, other adhesion and/or costimulatory lator:responder ratio of 1:4, higher stimulator:responder ratios molecules (which are also upregulated by CD40 triggering) may be needed to induce anergy. presumably reduce the B7 costimulatory threshold. Although Collectively, our findings indicate that B-CLL represents an the CD54 and CD58 adhesion molecules may importantly ideal candidate for further investigation of potential immuno- contribute to (antitumor) T cell responses,30–32 their strong therapeutic strategies since: (1) large numbers of tumor cells upregulation on CD40-triggered B-CLL cells was, however, can be easily obtained from small samples of peripheral insufficient to stimulate allogeneic T cells in the absence of blood; (2) freshly collected B-CLL cells can be maintained in B7 costimulation. In accordance with our findings, allogeneic culture for some weeks; (3) coculture of B-CLL cells with T cell proliferative responses against malignant B cells from CD40L-expressing cells results in a rapid endogenous upregul- non-Hodgkin’s lymphomas were also strongly inhibited by ation of adhesion and costimulatory molecules; and (4) both blocking CD80 and/or CD86 costimulatory molecules.23,33 autologous T cells and tumor cells can be easily purified by Most importantly, priming of allogeneic CD8+ T cells with immunomagnetic separation. The finding that CD40-trig- CD40-triggered B-CLL cells resulted in cytotoxic activity gering of B-CLL cells results in the functional expression of against the unstimulated B-CLL cells. Earlier observations indi- non-specific, costimulatory signals (signal 2) and strongly cated that B-CLL cells are resistant to anti-Fas mediated cyto- enhances their immunogenicity towards allogeneic T cells, toxicity34 and to lysis by normal or autologous LAK cells,35 stimulates further research to determine whether additional but our data show that they are vulnerable to CTL effector costimulatory factors may break the in vitro unresponsiveness mechanisms. On two occasions where we tried to generate of autologous T cells towards CD40-triggered tumor cells; CD40 triggering of chronic lymphocytic leukemia B cells LE Van den Hove et al 579 whether repetitive stimulation with CD40-triggered B-CLL 11 Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7 system of cells and cytokines may permit the detection of tumor-specific costimulation. Annu Rev Immunol 1996; 14: 233–258. autologous T cell clones; whether allospecific anergy can be 12 Chen L, Ashe S, Brady WA, Hellstro¨m I, Hellstro¨m KE, Ledbetter JA, McGowan P, Linsley PS. Costimulation of antitumor immunity induced in allogeneic T cells by coculture with resting B-CLL by the B7 counterreceptor for the T lymphocyte molecules CD28 cells at higher stimulator:responder ratios than used in the and CTLA-4. Cell 1992; 71: 1093–1102. present experiments; or whether B-CLL cells are deficient in 13 Townsend SE, Allison JP. Tumor rejection after direct costimu- the tumor antigen-specific signal (signal 1). These investi- lation of CD8+ T cells by B7-transfected melanoma cells. Science gations may help to define new strategies for the rational 1993; 259: 368–370. application of costimulation in tumor immunotherapy. 14 Baskar S, Glimcher L, Nabavi N, Jones RT, Ostrand-Rosenberg S. Major histocompatibility complex class II+ B7-1+ tumor cells are potent vaccines for stimulating tumor rejection in tumor-bearing mice. J Exp Med 1995; 181: 619–629. Acknowledgements 15 Matulonis UA, Dosiou C, Lamont C, Freeman GJ, Mauch P, Nadler LM, Griffin JD. Role of B7 in mediating an immune This work was supported by grants from the ‘Algemene Spaar- response to myeloid leukemia cells. Blood 1995; 85: 2507–2515. en Lijfrentekas’ (ASLK), Brussels; the ‘Onderzoeksfonds’ of the 16 Lanzavecchia A. Receptor-mediated antigen uptake and its effect on antigen presentation to class II restricted T lymphocytes. Annu Catholic University of Leuven (Grant OT 93/32); the ‘Fonds Rev Immunol 1990; 8: 773–793. voor Wetenschappelijk Onderzoek Vlaanderen’ (‘Krediet aan 17 Freedman AS. Immunobiology of chronic lymphocytic leukemia. 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