The Functional in Vitro Response to CD40 Ligation Reflects a Different

The functional in vitro response to CD40 ligation reﬂects a different clinical outcome in patients with chronic lymphocytic leukemia

This article has been corrected since advance Online Publication, and a corrigendum is also printed in this issue

C Scielzo1,2,9, B Apollonio1,3,4,9, L Scarfo` 5,6, A Janus6, M Muzio1,4, E ten Hacken3,6, P Ghia3,6,7,8 and F Caligaris-Cappio1,3,7,8

1Laboratory of Lymphoid Malignancies, Division of Molecular Oncology, Istituto Scientifico San Raffaele, Milan, Italy; 2Unversita` degli studi di Milano, Milan, Italy; 3Universita` Vita-Salute San Raffaele, Milan, Italy; 4Laboratory of cell activation and signalling, Division of Molecular Oncology, Istituto Scientifico San Raffaele, Milan, Italy; 5Haematology Section, University of Ferrara, Ferrara, Italy; 6Laboratory of B Cell Neoplasia, Division of Molecular Oncology, Istituto Scientifico San Raffaele, Milan, Italy; 7Unit of Lymphoid Malignancies, Department of Oncology, Istituto Scientifico San Raffaele, Milan, Italy and 8MAGIC (Microenvironment and Genes in Cancers of the blood) Interdivisional Research Program, Istituto Scientifico San Raffaele, Milan, Italy

Malignant B lymphocytes from chronic lymphocytic leukemia who have a poorer clinical outcome and tend to express other (CLL) patients maintain the capacity to respond to CD40 negative prognostic factors (for example, ZAP70 or CD38 ligation, among other microenvironmental stimuli. In this study, positivity, absence of IGHV gene mutations).6,8–10 we show that (i) leukemic CLL cells stimulated with the soluble form of CD40L in vitro show differential responses in terms of There is substantial evidence that CLL cells retain the capacity upregulation of surface markers (CD95 and CD80) and induc- to respond in vitro also to the so-called ‘second signal’ delivered tion of chemokines (CCL22 and CCL17) expression/secretion, through CD40,11 a member of the tumor necrosis factor receptor and that (ii) these changes are mirrored by a distinct activation family. In normal B cells stimulation by CD40-ligand (CD40L/ of intracellular signalling pathways including increase in CD154), the cognate ligand expressed on activated T lympho- IKKalpha/beta phosphorylation and upregulation of antiapop- cytes,12 occurs on Ag recognition and is crucial to initiate the totic proteins (BCL-2 and MCL-1). CLL patients can then be 13,14 segregated into two distinct functional subsets. We defined processes of B-cell proliferation and differentiation. In vitro the responsive subset of cases CD40L dependent, considering CD40 stimulation can rescue CLL cells from apoptosis and/or the capacity to respond as a sign of persistent need of this induce their proliferation;15–19 it modulates the expression of stimulation for the leukemic expansion. Conversely, we named surface molecules (for example, CD80; CD95)19–21 and of the unresponsive cases CD40L independent, considering them apoptosis-regulatory proteins such as BCL-2, survivin,17 BCL-X less dependent on this microenvironmental signal, presumably or MCL-1(refs 20–22) and the secretion of the chemoattractants because of a higher autonomous proliferative and survival CCL22 (MDC) and CCL17 (TARC).23 Conceivably, CD40 stimula- potential. Importantly, we report that (iii) the two functional þ þ subsets show an opposite clinical outcome, with CD40L- tion may occur also in vivo as CD4 CD40L T cells are independent cases having a shorter time to progression. This invariably present in infiltrated lymph nodes and bone marrow1,24 indicates that the functional differences observed in vitro may and tend to concentrate within tissue proliferation centers where reflect a different leukemic potential in vivo likely responsible proliferating malignant B cells cluster around them.25 for a distinct clinical course. The engagement of CD40 by CD40L induces activation of both Leukemia (2011) 25, 1760–1767; doi:10.1038/leu.2011.149; the canonical and non-canonical nuclear factor kB (NF-kB) published online 28 June 2011 26 Keywords: CLL; CD40L; prognosis signalling pathways. The activation of the canonical NF-kB pathway leads to the phosphorylation of the IKK complex including IKK alpha and IKK beta; the activation of non-canonical Introduction NF-kB signalling pathway involves specifically IKK alpha. There- fore, it is possible to analyze the phosphorylation status of IKK Survival and expansion of chronic lymphocytic leukemia kinases as a read-out of NF-kB signalling pathways activation.27 1,2 (CLL) clones are favored by stimuli originating from the Not all CLL clones respond to CD40 stimulation in vitro, 2 microenvironment. The stimulation through the B-cell receptor indicating a clonal functional heterogeneity.1,17,18 This is relevant 3,4 has been found to be a key factor, although the response to considering that CD40 has attracted great interest as a potential B-cell receptor stimulation is heterogeneous and only in a subset therapeutic target in CLL,28,29 and in ongoing clinical trials either of patients the malignant cells are responsive in vitro to the activation of CD40 by genetically modified leukemic cells immunoglobulin (Ig) crosslinking that leads to an effective cell expressing CD40L gene30 or CD40 inhibition with anti-CD40 5,6 activation. CLL cases unresponsive to Ig ligation show the antagonist antibody31 are used in different therapeutic strategies. functional and biochemical features of B cells anergized by an In this work, we have identified two distinct subsets of CLL in vivo antigen (Ag) encounter and a have a better clinical patients, which we have named CD40L dependent and CD40L 5,7 outcome as compared with CLL cases responsive to anti-IgM independent, based on the in vitro cell response to CD40 stimulation in terms of upregulation of surface molecules (CD95 Correspondence: Professor P Ghia, Laboratory of B Cell Neoplasia, and CD80), induction of chemokine expression (CCL22 and Division of Molecular Oncology, Universita` Vita-Salute San Raffaele, CCL17) or secretion (CCL22), as well as upregulation of c/o DIBIT 4A3, Via Olgettina 58, Milan 20132, Italy. antiapoptotic molecules (BCL-2 and MCL-1) and increased E-mail: [email protected] 9These authors equally contributed to this work. phosphorylation of signalling molecules (IKK alpha/beta). Received 20 January 2011; revised 6 April 2011; accepted 20 April Interestingly, CD40L-independent cases had a significantly worse 2011; published online 28 June 2011 clinical progression as compared with CD40L-dependent CLL. Response to CD40 ligation in CLL C Scielzo et al 1761 Methods DNA synthesis and PCR amplifications were carried out as described.39 Patients Leukemic lymphocytes were obtained from the peripheral blood Cell lysis and western blot analysis of 60 unselected CLL patients, diagnosed according to the 6 international guidelines.32 All tissue samples were obtained with After 5 and 30 min, 24 and 72 h of culture, 3 Â 10 were pelleted, approval of the institutional review board of San Raffaele Scientific resuspended in cold lysis buffer (150 mM NaCl, 1% NP40, 50 mM Institute (Milan, Italy). A total of 37 patients were untreated at the TrisHCl, 1 mM EDTA, protease inhibitors and phosphatase 1 time of the analysis among which eight cases progressed between inhibitors) and spinned in microcentrifuge at 4 C for 20 min at 16 and 217 months (median time to progression 61 months) after 13 000 r.p.m.; lysate was recovered from supernatant and 1 diagnosis. The remaining 29 remained stable for a median follow- immediately used for western blotting or frozen at À20 C. up of 62 months. Another 20 cases were studied after the first Whole-protein extracts from either fresh or thawed lysates progression and were off therapy for at least 6 months before the were resolved by sodium dodecyl sulfate-polyacrylamide gel beginning of the study. Three patients were lost to follow-up. The electrophoresis, and proteins from gel were electron transferred following parameters were analyzed for each patient: age, disease onto nitrocellulose membranes, and probed with anti-BCL2 stage at diagnosis according to Binet33 or modified Rai criteria,34 (Upstate Biotechnology, Billerica, MA, USA), anti-MCL-1, anti- CD38 expression,35 IGHV gene mutational status,36 ZAP-70 IKK alpha/beta (Santa Cruz Biotechnology, Santa Cruz, CA, expression,9 characterization of disease as progressive or stable USA), anti-phospho-IKK alpha/beta (Cell Signaling, Denvers, as defined by the IWCLL-NCIWG.32 MA, USA) or anti-beta-Actin (Sigma, St Louis, MO, USA) antibodies. Immunoreactivity was revealed by incubation with either goat anti-rabbit Ig, goat anti-mouse Ig (Upstate Biotech- Cell purification and in vitro CD40 stimulation nology) conjugated with HRP, followed by ECL reaction (Pierce, Mononuclear cells from all cases were isolated after Ficoll Waltham, MA, USA) and film exposure. Densitometric analysis gradient, and in a pilot series of 14 cases CD19 þ leukemic of protein-specific bands was performed using ImageQuant B cells were also purified using a B lymphocyte-enrichment kit Software (GE Healthcare, Upsala, Sweden). CLL MEC1 cell line 40 (RosetteSep; StemCell Technologies, Peschiera Borromeo, was used as positive control. Milan, Italy) and immediately used for the experiments depicted below. Purity of all preparations was always above 99% and the Enzyme-linked immunosorbent assay cells co-expressed CD19 and CD5 on their cell surfaces, as The presence of the chemokines CCL17 and CCL22 in the checked by flow cytometry (FC500; Beckman Coulter, Cassina supernatants from both untreated and CD40L-treated cells, De Pecchi, Milan, Italy); preparations were virtually devoid of cultured for 72 h, was detected and quantified by ELISA using NK, T lymphocytes and monocytes. commercially available kits (R&D Systems, Minneapolis, MN, Total peripheral blood mononuclear cells and purified USA). The lower limit of detection of the ELISA was 0.25 ng/ml B lymphocytes were resuspended in RPMI 1640 medium for CCL22 and 0.13 ng/ml for CCL17. (Life Technologies, San Giuliano Milanese, Milan, Italy), containing 10% fetal calf serum, 2 mML-glutamine and 15 mg/ml gentamicin, and cultured for up to 3 days with or without Statistical analysis soluble recombinant human CD40L (sCD40L) at 100 ng/ml plus Patients were grouped in two subsets (CD40L dependent and 1 mg/ml of enhancer, according to the manufacturer’s instruc- CD40L independent) according to their in vitro response to tions (Alexis Corporation, San Diego, CA, USA). sCD40L stimulation. Parametrical and non-parametrical data Cells were collected at different time points, for flow- were compared with the functional response (Table 1). For non- cytometryic analysis (24 and 72 h), western blotting experiments parametrical data, w2 test and Fisher exact test defined the (5 and 30 min for IKK and 24 and 72 h for BCL-2 and MCL-1), statistical significance (two-tales) of the contingency tables. For and RNA extraction (24 and 72 h). At each time point, culture parametrical data the analysis of variance test was used supernatants were collected, filtered and stored at À80 1C for (Table 1). Time to progression (TTP) was calculated from enzyme-linked immunosorbent assays (ELISA). diagnosis to first occurrence of progression according to IWCLL-NCI-WG criteria.32 TTP curves were plotted according to Kaplan–Meier method and compared with the log-rank test. Cytometric analyis A correlation was considered as statistically significant when the At 24 and 72 h, cultured cells were analyzed with a FC500 P value of the corresponding test was not greater than 5%. All cytometer (Beckman Coulter), acquiring at least 10 000 events statistical computations were conducted using SPSS version and using the following monoclonal antibodies: anti-CD19, 16.0 (SPSS, Chicago, IL, USA). Statistical analysis of western blot anti-CD20, anti-CD5, anti-CD38, anti-CD80 (Becton and data was performed using Student’s t-test. Dickinson, San Jose, CA, USA), anti-CD95 (Caltag Laboratories San Francisco, CA, USA)37 and ZAP70 expression was analyzed using the anti-ZAP70 antibody (Caltag, Burlingame, CA, USA) as Results previously described.38 In parallel, cell viability was also assessed using the Annexin V-based apoptosis detection kit CLL cells show a heterogeneous response to CD40L (R&D Systems, Flanders, NJ, USA). stimulation We cultured CLL B cells for up to 3 days in the presence of sCD40L and we evaluated changes in the expression of different RNA extraction and PCR molecules at protein and RNA level. After 24 and 72 h of culture, total RNA was extracted from First, we analyzed by flow cytometry the surface expression of 3 Â 106 cells using Trizol (Invitrogen life technologies, Paisley, the activation markers CD95 and CD80 that are upregulated UK), following the manufacturer’s instructions. Complementary following CD40 stimulation of normal B cells.21 At basal level,

Leukemia Response to CD40 ligation in CLL C Scielzo et al 1762 Table 1 Correlations with clinical and biological features

CD40 L dependent CD40 L independent P-value

Median age at diagnosis 64 (44–82) 61 (43–76) 0.364a CD38 positive (X20%) 35.3% (12/34) 19.2% (5/26) 0.249a Unmutated IGHV genes 26.5% (9/34) 36.0% (9/25) 0.569a ZAP70 positive (X20%) 31.6% (6/19) 71.4% (10/14) 0.037a Disease progression 15.6% (5/32) 40% (10/25) 0.067a LDTo6 month 11.1% (2/18) 28.6% (4/14) 0.365a LDT mean rate 41.1% 66.62% 0.332b Need for therapy 37.5% (12/32) 64.0%(16/25) 0.064a Modiﬁed Rai’s stage Low 62.5% (20/32) Low 70.8% (17/24) 0.801a Intermediate 31.2% (10/32) Intermediate 25.0% (6/24) High 6.2% (2/32) High 4.2% (1/24) b2microglobulin 2.70 mg/dl 4.07 mg/dl 0.31b Mean lymphocyte count 34 660/mm3 65 010/mm3 0.012b Mean hemoglobin serum levels 13.16 g/dl 12.49 g/dl 0.334b Mean platelets count 153 270/mm3 170 700/mm3 0.480b Follow-up median time (months) 85 81 0.628b Time to progression (months) 168 78 0.041c Abbreviation: LDT, lymphocyte doubling time. aP-values for the comparison between CD40L-dependent and -independent cases were calculated using Fisher’s exact test. bP-values refer to one-way ANOVA test. cLog-rank test.

Figure 1 Analysis of CLL cells response to sCD40L stimulation. (a) The graphs show the percentage of CD80 (top panels) and of CD95 (bottom panels) increase by leukemic cells after in vitro culture in the presence of sCD40L for each individual case. (b) The gels show CCL17 expression by RT-PCR in leukemic cells from one CD40L-dependent (upper panel) and one CD40L-independent (lower panel) patients cultured for 24 and 72 h with ( þ ) or without (À) sCD40L. T0 shows the level of chemokine expression at basal level. b-actin (lower panels) was used as positive control. (c) Graphs show the amount of secreted CCL22/MDC and CCL17/TARC after CD40L stimulation at different concentration, evaluated by ELISA.

CD95 expression was absent in 59/60 CLL cases, while CD80 46 patients. Although by reverse-transcription PCR we observed a was detected in 27/60 cases (45%). After CD40 stimulation, significant increase in gene expression only for CCL17 (Figure 1b) malignant cells from 34/60 patients (56.7%) showed the (27/46), in all these cases the secretion of both chemokines was induction of CD95 and/or the upregulation of CD80 expression actually increased as detected by ELISA (Figure 1c). (Figure 1a). In addition, the same cases showed also an increase In 27/46 (58.7%) cases we observed a concomitant functional in cell viability of 26±9% as compared with control cultures response after CD40 stimulation with all the four assays utilized (data not shown).18 (cytofluorimetric analysis, reverse-transcription PCR assay for Next, we evaluated the expression and secretion of the CCL17, ELISA for CCL22 and ELISA for CCL17). On the basis of chemokines CCL22 and CCL17 in response to CD40 ligation in these results we defined these cases CD40L dependent.

Leukemia Response to CD40 ligation in CLL C Scielzo et al 1763 The remaining 21 patients (45.7%) did not show any and 12 CD40L dependent) for BCL-2 expression at basal level phenotypic and functional modifications after in vitro CD40 showed that the levels of expression before stimulation were stimulation (14 cases) or had marginal changes in only one of associated with the capacity to respond to CD40L stimulation in the four assays (one CD80/CD95 upregulation, one induction of vitro (P ¼ 0.0019) (densitometric analysis Figure 3c) suggesting a PCR band for CCL17, six positive ELISA assays for CCL22). All that the expression of this antiapoptotic molecule may depend these cases were defined CD40L independent. on a similar stimulation also in vivo. To exclude that T, NK and monocytes cells could affect Finally, in four patients (two CD40L dependent and two CD40L differential response in vitro, we performed parallel CD40L-independent), we also evaluated MCL-1 expression, and stimulation experiments using unfractioned peripheral blood similarly to BCL-2, we observed a significant protein increase mononuclear cells and purified leukemic B cells from 14 cases (seven dependent and seven independent). We did not observe any differences in terms of response confirming, as expected, the cell specificity of the stimulation through CD40 receptor (data not shown). Positive control To + CD40L (5’) + CD40L (30’) A biochemical signature for CD40 response α β β On the basis of these results, we aimed at defining biochemical pIKK / α features that might correlate with the different functional response and help distinguishing the two subsets of patients. IKK α This set of experiments was performed only on purified leukemic B cells (99% pure) from the peripheral blood of CLL CD40L-Dependent patients, after culture in the presence or the absence of sCD40L. CD40L response was evaluated in 12 CLL samples (six CD40L α β β dependent and six CD40L independent) at early time points pIKK / α (5 and 30 min after stimulation) by analyzing the phosphorylation status of IKK alpha/beta by western blotting. As shown in Figure 2a, only CD40L-dependent patients (6/6) showed a IKK α significant upregulation of phospho-IKK alpha/beta as compared with the untreated samples, while CD40L-independent patients CD40L-Independent (6/6), (Figure 2a), showed no significant enhancement in IKK alpha/beta phosphorylation (see Figure 2b for comparison 1500 between the two subsets). In addition, we observed that the basal levels of IKK alpha/beta phosphorylation are different among the two groups, with CD40L-independent cases showing ** P=0.0087 a higher level of basal activation (P ¼ 0.0279) (Figure 2c). We β / 1000 then evaluated by western blotting the levels of protein α expression at later time points (24 and 72 h) after sCD40L /IKK β treatment, focusing our attention on the antiapoptotic molecules / BCL-2 and MCL-1. We tested BCL-2 expression in six CD40L- α dependent and six CD40L-independent cases and we observed 500 that the level of the protein in cells from CD40L-dependent % pIKK patients was significantly upregulated (P ¼ 0.04) in contrast to CD40L-independent samples (Figures 3a and b). Interestingly, the analysis of 23 further CLL patients (11 CD40L independent 0

CD40L CD40L Independent Dependent Figure 2 Early response to sCD40L stimulation. Leukemic B cells 200 from 12 patients (six CD40L dependent and six CD40L independent) were puriﬁed and put in culture with sCD40L for 5 and 30 min. (a) The western blot analysis shows IKK alpha/beta phosphorylation as * P=0.0279 compared with IKK alpha/beta basal level. Two representative β experiments are shown from one CD40L-dependent (upper panels) / α 100 and one CD40L-independent patient (lower panels). The different molecular weights of IKK alpha (85 kDa ¼ a) and IKK beta (87 kDa ¼ b /IKK β (upper panel)) are shown on the right side of the blots. / α (b) Densitometric clustered analysis of phospho-IKK alpha/beta increase at 30 min after sCD40L stimulation in six CD40L-independent 0

(left panel) and six CD40L-dependent patients (right panel). Phospho-IKK % pIKK alpha/beta increase was expressed as the difference between (phospho-IKK alpha/beta)/(IKK alpha/beta) optical density after stimulation and (phospho-IKK alpha/beta)/(IKK alpha/beta) optical density in the untreated sample. (c) Basal phospho-IKK alpha/beta protein levels -100 were analyzed by western blot and the graph shows the levels as CD40L CD40L quantiﬁed by densitometric analysis (11 independent and 11 Independent Dependent dependent patients).

Leukemia Response to CD40 ligation in CLL C Scielzo et al 1764

Figure 3 CD40L long-term response. Purified CLL cells from patients were stimulated with CD40L or left untreated for 24 and 72 h. Cells were harvested, lysed and resolved by western blot analysis for BCL-2 (a–c) or MCL-1 (d, e). (a) BCL-2 protein levels were analyzed after cell purification (To) and in untreated cells (À) and in sCD40L-stimulated samples ( þ ) after 24 and 72 h. Two representative patients, one CD40L independent (top panels) and one CD40L dependent (lower panels), are shown. MEC1 cells were used as positive control. (b) BCL-2 protein levels were quantified by densitometric analysis and are expressed as the ratio between BCL-2 bands optical density (BCL-2 OD) and b-actin bands optical density (b-actin OD). (c) BCL-2 protein levels were investigated by western blot and quantified by densitometry at basal level (11 independent and 12 dependent patients). (d) MCL-1 protein levels were analyzed after cell purification (To) and in untreated cells (À) and in sCD40L-stimulated samples ( þ ) after 24 and 72 h both. Two representative patients one CD40L independent (top panels) and one CD40L dependent (lower panels), are shown. MEC1 cells were used as positive control. (e) MCL-1 protein levels were quantified by densitometric analysis and are expressed as the ratio between MCL-1 bands optical density (MCL-1 OD) and b-actin bands optical density (b-actin OD). Two representative patients one CD40L independent (top panel) and one CD40L dependent (bottom panel), are shown, at 24 and 72 h, with or without sCD40L.

only in the CD40L-dependent cases at both 24 and 72 h as response to CD40 ligation was able to further subdivide these compared with controls (Figures 3d and e). patients in terms of clinical outcome and prognosis (Figure 4b). In this patient sub-population all CD40L-independent cases had a progressive disease (5/5, 100%), while the majority of CD40L- Clinical correlations dependent cases (8/11, 72.7%) had a stable disease (P ¼ 0.026). To assess whether the observed functional difference might have Among CD38 þ cases, TTP was also significantly shorter in clinical implications, we retrospectively correlated several CD40L-independent patients (20 months vs 130 months, clinical parameters (Table 1) with the in vitro response to P ¼ 0.001). CD40 ligation. CD40L-dependent cases had a lower mean lymphocyte count in peripheral blood in comparison with CD40L independent (34 660/ml vs 65 010/ml, P ¼ 0.012, at the Discussion time of analysis while hemoglobin, platelet and b2-microglobulin values were similar in both groups. Although the CD40/CD40L interactions are crucial for maturation, expansion differences between the two groups in terms of disease and survival of normal B lymphocytes. Malignant B cells make progression and need for treatment did not reach statistical no exception as leukemic CLL B cells maintain the capacity to significance (P ¼ 0.067 and P ¼ 0.064, respectively), TTP curves respond to several microenvironmental stimuli including CD40 showed a clear advantage for CD40L-dependent patients, who ligation.25 Here we show that (i) CLL B cell clones may show had a median TTP of 168 months compared with the 78 months different responses in terms of upregulation of surface markers of of CD40L independent (P ¼ 0.041) (Figure 4). activation (CD95 and CD80) and induction of chemokines A significant association was observed only with ZAP70 (CCL22 and CCL17) expression/secretion and that (ii) these expression (P ¼ 0.0037) among the major prognostic markers changes are mirrored by a distinct activation of intracellular (CD38, IGHV gene mutations, see Table 1). Interestingly, when signalling pathways including increase in IKK alpha/beta we analyzed the CD38-positive subgroup (16 patients), the phosphorylation as well as an upregulation of antiapoptotic

Leukemia Response to CD40 ligation in CLL C Scielzo et al 1765 distinct functional subsets show an opposite clinical outcome, 1.0 CD40L Dependent with CD40L-independent cases having a shorter TTP (Figure 4a; CD40L Independent Table 1). This indicates that the functional differences observed 0.9 in vitro may reﬂect a different leukemic potential in vivo that may be responsible for a distinct clinical course. 0.8 Such heterogeneity of response has not been observed when CD40L was used to increase immunogenicity of CLL cells, by stably transducing CD40L either in stromal cells42 or directly in 0.7 CLL cells30,43 to crosslink CD40 on transduced and non- transduced bystander CLL cells. Conceivably, the strength

Cum Survival 0.6 of the signal delivered to the leukemic cells was higher than that provided by the soluble form of CD40L that we have used in this work. The high levels of expression achieved after 0.5 transduction of molecules in a membrane-bound form likely produce a much stronger effect that, although beneficial when 0.4 the purpose is to increase the leukemic cells immunogenicity, could not adequately represent the currently unknown intensity 0 50 100 150 200 250 of the signal occurring in vivo. Along the same line of reasoning, TTP we have previously reported that the use of 10 times higher Log Rank (Mantel-Cox): 0.041 concentrations of soluble CD40L could indeed obtain a Breslow (Generalized Wilcoxon): 0.035 response from all cases, although the level of response in terms of cellular modifications was still different in different subsets of patients.44 1.0 Patients CD38>20% We have observed a significant correlation between the type of response to CD40 ligation (dependent vs independent) and the clinical parameters of poor outcome, including ZAP70 0.8 expression, while we were unable to find any correlation with other classical ‘biological’ prognostic markers like CD38 expression35 or IGHV gene mutational status36 as one might 0.6 have expected. Recently, correlations between functional features of CD40 ligation and IGHV gene mutational status have been reported in some instances.42,45 This discrepancy 0.4 could be simply explained by the different number of cases Cum Survival tested and/or by differences in the experimental models used, as CD40 stimulation was studied together with additional stimuli 0.2 like cytokines45 or Toll-like receptors ligands.42 A final answer CD40L Dependent will be only obtained with a prospective study involving 0.0 CD40L Independent hundreds of cases to eliminate any selection bias. Such a study will also help elucidating whether the capacity to respond to 0 50 100 150 200 CD40 stimulation remains stable over time and can predict the TTP outcome at any given moment during the disease course or whether it modifies indicating a functional switch that may Log Rank (Mantel-Cox): 0.001 herald a clinical turning point. Breslow (Generalized Wilcoxon): 0.003 It is known that poor prognosis cases (generally defined by, þ þ Figure 4 Clinical correlations. (a) Kaplan–Meyer estimated curves of but not limited to, IGVH unmutated cases, ZAP70 or CD38 progression probability. Time to progression of CLL patients classified cases or cases expressing specific stereotyped receptors) tend to into two groups according to CD40L response: (1) CD40L-dependent maintain the capacity to be stimulated through the B-cell subjects (n ¼ 34); (2) CD40L-independent subjects (n ¼ 26). Results of receptor usually being induced to enter the cell cycle. In the statistical analysis are shown below the plot. (b) TTP of CLL contrast, we here show that poor prognosis cases do not likely X patients positive for CD38 expression (CD38 expression 20%) respond to CD40 ligation, suggesting the independence from the stratified according to CD40L response: (1) CD40L dependent (n ¼ 12); (2) CD40L-independent (n ¼ 5). Results of the statistical need of a classic T-cell help. This is also in keeping with analysis are shown below the plot. previous experimental evidences where stimulation with CpG- DNA showed a heterogeneous response in CLL cells with the progressive ones being induced to proliferate.46,47 This dual proteins (BCL-2 and MCL-1). Based on the level of activation B-cell receptor–Toll-like receptor engagement (in the absence of achieved by leukemic B cells when stimulated with the soluble CD40 stimulation) is reminiscent of the classical model of form of CD40L in vitro, CLL patients can segregate into two thymus-independent type I antibody responses in autoimmune distinct functional subsets. We defined the responsive subset of disorders48 and could then be hypothesized to occur also in the cases CD40L dependent, considering the capacity to respond as natural history of at least a subset of CLL, especially with a sign of persistent need of this stimulation that is available progressive disease. in vivo for the leukemic clonal expansion.41 Conversely, we Along the same line, we were able to show that the response named the unresponsive cases CD40L independent, considering to sCD40L significantly discriminated the clinical outcome them less dependent on this microenvironmental signal, among CD38 þ patients (Figure 4b), with 8/11 CD38 þ , CD40L- presumably because of a higher autonomous proliferative and dependent patients being stable while all (5/5) CD38 þ , CD40L- survival potential. The relevant observation is that the two independent progressed (P ¼ 0.0026).

Leukemia Response to CD40 ligation in CLL C Scielzo et al 1766 In conclusion, based on in vitro cellular and biochemical 11 Liu YJ, Joshua DE, Williams GT, Smith CA, Gordon J, MacLennan IC. responses following ligation with soluble CD40 ligand, we have Mechanism of antigen-driven selection in germinal centres. Nature identified two distinct subsets of CLL patients (named CD40L 1989; 342: 929–931. dependent and CD40L independent). Interestingly, CD40L- 12 Stamenkovic I, Clark EA, Seed B. A B-lymphocyte activation molecule related to the nerve growth factor receptor and induced independent cases had a worse clinical outcome as compared by cytokines in carcinomas. EMBO J 1989; 8: 1403–1410. with CD40L-dependent CLL, in terms of clinical progression. 13 van Kooten C, Banchereau J. Functions of CD40 on B cells, The existence of two distinct functional subsets that correlate dendritic cells and other cells. Curr Opin Immunol 1997; 9: with a distinct outcome, confirm the role of the microenviron- 330–337. ment and its stimuli in the natural history of CLL, where the 14 Kehry MR. CD40-mediated signaling in B cells. Balancing cell activation of selected intracellular pathways may become survival, growth, and death. J Immunol 1996; 156: 2345–2348. 15 Buske C, Gogowski G, Schreiber K, Rave-Frank M, Hiddemann W, responsible for specific disease courses. Wormann B. Stimulation of B-chronic lymphocytic leukemia cells by murine fibroblasts, IL-4, anti-CD40 antibodies, and the soluble CD40 ligand. Exp Hematol 1997; 25: 329–337. Conflict of interest 16 Furman RR, Asgary Z, Mascarenhas JO, Liou HC, Schattner EJ. Modulation of NF-kappa B activity and apoptosis in chronic The authors declare no conflict of interest. lymphocytic leukemia B cells. J Immunol 2000; 164: 2200–2206. 17 Granziero L, Circosta P, Scielzo C, Frisaldi E, Stella S, Geuna M et al. 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