Blood dendritic cells in patients with chronic lymphocytic leukaemia

ARTICLE IN PRESS

Immunobiology 213 (2008) 493–498 www.elsevier.de/imbio

SHORT COMMUNICATION

Blood dendritic cells in patients with chronic lymphocytic leukaemia Naira Ben Mamia,b, Mohamad Mohtya,b,c,d, The´re`se Aurran-Schleinitzc, Daniel Olivea,b, Be´atrice Gauglera,b, a

Laboratoire d’Immunologie des Tumeurs, Institut Paoli-Calmettes, Universite´ de la Me´diterrane´e, 232 Bd. Ste. Marguerite, 13273 Marseille, Cedex 09, France b Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) UMR 599, 232 Bd. Ste Marguerite, 13273 Marseille, France c De´partement d’He´matologie, Institut Paoli-Calmettes, Marseille, France d Unite´ de Transplantation et de The´rapie Cellulaire, Institut Paoli-Calmettes, Marseille, France Received 13 July 2007; received in revised form 18 November 2007; accepted 22 November 2007

Abstract Myeloid and plasmacytoid dendritic cells (MDC, PDC) play a key role in the initiation of immune responses. We found a reduction of both DC subsets in 42 patients with chronic lymphocytic leukaemia (CLL) at diagnosis (Po0.0001 and 0.0001 vs. controls, respectively), likely related to the high secretion of CCL22 and CXCL12 (P ¼ 0.04 and 0.008 vs. controls, respectively) by leukaemic cells. However, CD14+ monocytes from CLL patients could give rise to functional IL-12p70-secreting monocyte-derived DCs, capable of inducing a type 1 polarization immunostimulatory profile. These monocyte-derived DCs from CLL patients efficiently migrate in response to CCL19/MIP-3b chemokine, suggesting that functional autologous DCs can be generated for immunotherapeutic purposes to circumvent DC defects in CLL. r 2007 Elsevier GmbH. All rights reserved. Keywords: Chronic lymphocytic leukaemia; Dendritic cells; Immunotherapy

Introduction Dendritic cells (DCs) are responsible for the initiation and regulation of immune responses. At least two subsets of blood DCs have been characterized based on the differential expression of CD11c. Myeloid DCs (MDC) and plasmacytoid DCs (PDC) can promote polarization of naive T cells (Cella et al., 2000; Rissoan et al., 1999) and induction of an efficient immune response. Thus, DC Corresponding author at: Institut National de la Sante´ et de la

Recherche Me´dicale (INSERM) UMR 599, 232 Bd. Ste Marguerite, 13273 Marseille, Cedex 09, France. Tel.: +33 491 223641; fax: +33 491 223610. E-mail addresses: [email protected] (M. Mohty), [email protected] (B. Gaugler). 0171-2985/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2007.11.009

functional defects may play a key role in tumour immune escape, and restoration of DC functions is a logical target for immunotherapy. In this study, we investigated the status of circulating DCs in the blood of 42 chronic lymphocytic leukaemia (CLL) patients. In addition, we tested the ability of CD14+ monocytes from CLL patients to give rise to functional DCs in vitro.

Patients, materials and methods Patients and controls CLL cell samples were obtained after informed consent from 42 patients at diagnosis. CLL diagnosis was

ARTICLE IN PRESS 494

N.B. Mami et al. / Immunobiology 213 (2008) 493–498

performed at the Institut Paoli-Calmettes (Marseille, France) according to standard criteria (Table 1). Peripheral blood mononuclear cells (PBMC) from patients and healthy controls (Etablissement Franc¸ais du Sang, Marseille, France) were separated on a FicollHypaque (Pharmacia, Uppsala, Sweden) density gradient. Plasma fraction was stored at 80 1C for later use. Normal, and leukaemic MDCs and PDCs were identified by three-color staining as previously described (Mohty et al., 2001). Plasma monocyte-derived chemokine (CCL22) and chemokine ligand CXCL12/SDF-1 levels were determined using specific ELISA (R&D Systems, Abingdon, UK).

In vitro DC generation CD14+ monocytes from healthy donors and CD14+ monocytes (as determined by FACS analysis in peripheral blood) from CLL patients were immunomagnetically purified with CD14 mAb-conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and were cultured with GM-CSF and IL-4 as previously reported (Mohty et al., 2003). Final maturation of monocyte-derived DCs was induced by adding 75-Gy irradiated CD40L-transfected murine Lcells (2  105 well). Supernatants of DC cultures were harvested at day 5, and at day 7 after 2 days of maturation with CD40L. IL-10, IL-12p70 and TNF-a concentrations were measured using specific ELISA with the OptEIA sets (BD-Biosciences, Le Pont de Claix, France). Quantification was normalized to 106 cells.

Flow cytometry analysis The following mAb were used for flow cytometry: anti-CD1a, CD14, CD80, CD83, HLA-DR and relevant isotypic controls, all from Beckman–Coulter (Marseille, France). CD86 was from BD-Biosciences. Samples were analysed on a FACSCanto cytometer and data were analysed using BD-FACSDiva software (BD-Biosciences).

T cell separation and mixed lymphocyte reaction (MLR) CD4+ T cells were purified by depletion of adult blood PBMC using the CD4 Cell Isolation Kit II (Miltenyi Biotec). CD4+/CD45RA+ naı¨ ve T lymphocytes were immunomagnetically isolated from the CD4+ T fraction by CD45RA mAb-conjugated microbeads (Miltenyi Biotec). CD8+ T lymphocytes were immunomagnetically purified from PBMCs by positive selection using CD8 mAb-conjugated microbeads (Miltenyi Biotec). T cell proliferation capacity was

Table 1.

Patients’ characteristics

UPN

Age

Sex

Disease stagea

% CD5+/CD19 + leukaemic cellsb

CLL1 CLL2 CLL3 CLL4 CLL5 CLL6 CLL7 CLL8 CLL9 CLL10 CLL11 CLL12 CLL13 CLL14 CLL15 CLL16 CLL17 CLL18 CLL19 CLL20 CLL21 CLL22 CLL23 CLL24 CLL25 CLL26 CLL27 CLL28 CLL29 CLL30 CLL31 CLL32 CLL33 CLL34 CLL35 CLL36 CLL37 CLL38 CLL39 CLL40 CLL41 CLL42

67 80 84 47 76 75 58 55 76 42 61 71 82 51 45 67 49 51 52 66 49 75 35 77 76 69 66 62 36 70 42 64 65 56 73 65 70 44 59 61 65 67

M F F M M M F F M M F M M M M F F F M M M F M F F F M M M F F M F M M F M M F F F F

C C C B A C C A A A A C A A A B A C A A A A A A B C A C A A A B A A A A A A B C A A

93 94 45 43 72 94 94 80 90 77 84 97 91 72 71 75 70 85 71 95 78 86 89 61 91 84 94 89 74 85 63 98 71 83 91 62 64 30 54 85 50 59

Abbreviations: F, female; M, male; CLL, chronic lymphocytic leukaemia; NA, not available. a Disease staging was performed according to the Binet classification. b As determined by flow cytometry in the peripheral blood.

evaluated as previously described (Mohty et al., 2003). Allogeneic CD4+CD45RA+ T cells or CD8+ T cells (105 cells/well) were co-cultured with DC (104 cells/well), cells were harvested after 7 days and replated in 96-well culture plates at 104 cells per well in triplicate in the presence of 25 ng/ml PMA (Sigma, St. Louis, MO) and

ARTICLE IN PRESS N.B. Mami et al. / Immunobiology 213 (2008) 493–498

1 mg/ml ionomycin (Sigma). After 24 h, supernatants were harvested. Cytokines were analysed by ELISA using the OptEIA Human set for IFN-g. Neutralization experiments were performed by adding mAbs against IL-10 (R&D Systems, 0.25 mg/ml) and IL-10-Receptor (R&D Systems, 1 mg/ml) at time of DC maturation and at the time of co-culture with CD4+ naive lymphocytes or CD8+ lymphocytes.

Migration assay To evaluate migration of DCs, the transwell system with 6.5 mm polycarbonate filters of 8-mm pore size was used (Costar, Cambridge, UK). DCs (105 cells) were suspended in 0.5 ml AIMV medium (Invitrogen Corporation, Auckland, New-Zealand) and added to the upper compartment. The lower compartment of the chamber was filled with 0.5 ml of AIMV medium containing 300 ng/ml of MIP-3b/CCL19 (R&D Systems). After 3 h of incubation at 37 1C, in 5% CO2, the content of the lower compartment was collected for DC counting using the FACSCanto cytometer.

Statistical analysis The significance of the differences between patients and controls was evaluated with the Mann–Whitney U-test. A P-value of 0.05 or less was considered to represent significance.

Results and discussion Blood DC subsets were analysed by flow cytometry after staining with lineage markers, CD11c and ILT3 (Mohty et al., 2001). CLL samples (Table 1) showed a significant reduction in the proportions of both MDCs and PDCs in comparison to healthy donors (Po0.0001 and 0.0001, respectively; Fig. 1A). Intriguingly, plasma from CLL patients contained significantly higher levels of CCL22 (Fig. 1B) and CXCL12 (Fig. 1C), two chemokines that were already shown to be produced by tumour cells (Zou, 2005). In order to circumvent blood DC depletion in CLL, we investigated the DC-generating capacity of circulating CD14+ monocytes isolated from CLL. After culture in standard conditions with GM-CSF and IL4, CD14+ monocytes isolated from CLL could give rise to genuine functional monocyte-derived DCs (Figs. 1D and E), capable of secreting significant amounts (although slightly lower than that of mature normal DCs; P ¼ NS) of bioactive IL-12p70 after maturation with CD40L (Fig. 1G). In addition, T cell proliferation

495

in response to monocyte-derived DCs from CLL patients was not statistically different from that in response to DCs from healthy donors (Fig. 1F). In accordance with IL12-p70 secretion, both CD4+ and CD8+ T cells cultured with these mature monocyte-derived DCs secreted significant amounts of IFN-g, but little or undetectable IL-10 and IL-4 comparable to healthy donors (Figs. 1(H) and (I) and data not shown), suggesting that monocyte-derived DCs can induce a type 1 polarization immunostimulatory profile. Finally, mature monocyte-derived DCs could efficiently migrate in response to the MIP-3b chemokine (CCL19; Fig. 1J), further confirming that functional autologous DCs can be generated in vitro from CLL patients for immunotherapeutic purposes. Although blood DCs might not completely reflect the status of DCs in other tissues, potential consequences can arise from the above observations. DC development in CLL is significantly affected by the leukaemic process and may contribute to immune dysregulation (Scrivener et al., 2001). While it is tempting to speculate that DC status may correlate with CLL disease stage (more alterations in advanced disease stage), we were unable to confirm such hypothesis likely due to a relatively low number of patients analysed from the advanced stages. Tumour-derived chemokine CXCL12 can mediate the trafficking of DCs into the tumour site, likely lymph nodes in CLL. Tumour DCs can induce IL-10+CCR7+ suppressive CD8+ T cells (Zou et al., 2001), which can inhibit tumour-specific central priming. The chemokine CCL22 derived from tumour cells and associated macrophages mediates CD4+CD25+ FOXP3+ regulatory T cell trafficking into the tumour site, as already shown in ovarian carcinoma (Curiel et al., 2004). At the therapeutic level, and though we did not assess the capacity to kill autologous CLL cells, our demonstration of the capacity of CD14+ monocytes derived from CLL to give rise to functional DCs in vitro suggests that DC-based vaccination approaches are attractive, especially at early disease stages, because one of the mechanisms by which CLL cells evade killing by the immune system is the impairment of DC functions (Goddard et al., 2001; Orsini et al., 2003). The optimal conditions (yield, clinical grade culture and maturation conditions, etc.) for DC generation from CLL patients are yet to be established. Nevertheless, ex vivo-generated DCs from CLL already proved to be capable of restoring crucial immune functions both in vitro and in vivo (Goddard et al., 2003; Hus et al., 2005). Thus, one could reasonably hypothesize that the application of DC vaccinations may help in overcoming the host immune tolerance against tumour cells for better long-term leukaemic immune control.

ARTICLE IN PRESS 496

N.B. Mami et al. / Immunobiology 213 (2008) 493–498

Acknowledgements

sur le Cancer (ARC)’’, the ‘‘Ligue Nationale contre le Cancer’’, the ‘‘Fondation de France’’, the ‘‘Fondation contre la Leuce´mie’’, the ‘‘Agence de Biome´decine’’, the ‘‘Association Cent pour Sang la Vie’’, and the ‘‘Association Laurette Fuguain’’ for their generous and continuous support for our clinical and basic research work.

We thank J. Wolfers (Immunotech, Beckman-Coulter, Marseille) for kindly providing the ILT3mAb. We thank N. Baratier and S. Just-Landi for excellent technical assistance. We also thank the ‘‘Association pour la Recherche

3500

2,00 P < 0.0001

3000

P < 0.0001

1,60 CCL22 (pg/ml)

% of PBMC

1,40 1,20 1,00 0,80 0,60 0,40

1000

P = 0.04

2500

CXCL12 (pg/ml)

1,80

1200

2000 1500 1000

800 600 400 200

500

0,20 0,00 MDC

0

PDC

P = 0.0008

Healthy donors

CLL

CD83

HLA-DR

0

Healthy donors

CLL

4500 CD14

CD86

CD80

Immature normal DC

4000

Mature normal DC

3500

Immature leukemic DC

3000

CD1a

MFI

Imm.

Mature leukemic DC

2500 2000 1500 1000

Mat.

500 0 CD80

CD86

CD83

HLA-DR

12000

15000

10000

Mature normal DC Immature leukemic DC 10000

Matureleukemic DC

5000

IL-12p70 (pg/ml)

(3H) Thymidine incorporation

Immature normal DC

8000 6000 4000 2000

0

0

10000

3000 1000 Stimulating cells

300

0

al

DC

al

.n

m

Im

ic

rm

m

or

DC

M

. at

DC

ic

em

no

em

uk

uk

e .l

m

Im

e .l

at

M

DC

ARTICLE IN PRESS N.B. Mami et al. / Immunobiology 213 (2008) 493–498

IFN-gamma (pg/ml)

20000

14000

Healthy donors

12000

CLL patients

IFN-gamma (pg/ml)

24000

16000 12000 8000 4000

497

Healthy donors CLL patients

10000 8000 6000 4000 2000 0

0 Imm. DC

Mat. DC

Imm. DC

Mat. DC

250 Immature normal DC

Number of DCs

200 150

Mature normal DC Immature leukemic DC Mature leukemic DC

100 50 0 -MIP-3 beta

+ MIP-3 beta

Fig. 1. (Continued)

Fig. 1. Status of circulating DCs in CLL patients. (A) Comparison of MDCs and PDCs % between healthy donors and CLL patients. PBMCs isolated from 15 healthy volunteers (black symbols) and 20 CLL patients (white symbols) were analysed by flow cytometry after three-colour staining with a combination of FITC-labelled monoclonal antibodies against lineage markers (CD3, CD14, CD16, CD19 and CD56), PE-labelled anti-CD11c and PC5-labelled anti-ILT3. Two distinct populations of lin-/ILT3+ cells were observed with respect to the expression of CD11c with the phenotypes of lin-/CD11c+/ILT3+ (MDC) and lin-/CD11c-/ILT3+ (PDC), (-) indicates median value. (B) CCL22 concentration determined by ELISA in the serum of 4 healthy donors and 17 CLL patients from this series, (-) indicates median value. (C) CXCL12 concentration determined by ELISA in the serum of four healthy donors and 17 CLL patients from this series. (D) Phenotype of monocyte-derived DCs generated in vitro from CLL patients. After culture with GM-CSF and IL-4, CD14+ monocytes from CLL patients gave rise to immature CD1a+/CD14 DCs (upper panel), that can be further matured after exposure to CD40L as shown by the up-regulation of CD80, CD86, CD83 and HLA-DR (lower panel). Data from one representative experiment are provided. Vertical bars denote the isotype control. (E) Histograms represent the mean fluorescence intensities of specific staining of the indicated cell surface marker (data from CLL26, CLL28, CLL35 and CLL42 analysed in four independent experiments). (F) Allostimulatory capacity of DCs generated from CLL patients. CD4+/CD45RA+ naive T cells were purified by negative selection of adult blood PBMC. Serial dilutions (1  104–3  102 cells/well) of stimulating cells were cultured in triplicate with 105 allogeneic naive T cells in 96-well flat bottom plates. Proliferation of T cells was monitored by measuring methyl-[3H] thymidine incorporation during the last 16 h of a 6-day culture. Normal DCs were used as control stimulators. The mean results obtained from CLL5, CLL23, CLL25, CLL26 and CLL28 in five independent experiments are indicated. (G) The IL-12p70 secretion profile of monocyte-derived DCs from CLL. Culture supernatants from 106 normal DCs and monocyte-derived DCs from CLL were harvested at day 7 after culture (after 48 h of exposure to CD40L). Cytokine secretion was analysed by ELISA. Results are represented as the mean and SEM values obtained from nine healthy donors and seven CLL patients (CLL5, CLL23, CLL25, CLL26, CLL28, CLL35 and CLL42). (H, I) T cell-polarizing capacity of monocyte-derived DCs in CLL. IFN-g content of supernatants from a 7-day co-culture of (H) CD4+/CD45RA+ naive T cells or (I) CD8+ T cells with the indicated stimulating cells was measured by ELISA after 24 h of PMA and ionomycin stimulation. Results are represented as the mean and SEM values of four independent experiments (CLL31, CLL35, CLL39 and CLL42). (J) Migration capacity of monocyte-derived DCs in CLL. 105 DCs were placed in the upper well of the Transwells plate for 3 h with or without MIP-3b (300 ng/ml). DCs counts in the lower well were measured by FACSCanto. Normal DCs were used as control. Results are represented as the mean and SEM values of four independent experiments (CLL17, CLL30, CLL31 and CLL35).

ARTICLE IN PRESS 498

N.B. Mami et al. / Immunobiology 213 (2008) 493–498

References Cella, M., Facchetti, F., Lanzavecchia, A., Colonna, M., 2000. Plasmacytoid dendritic cells activated by influenza virus and CD40L drive a potent TH1 polarization. Nat. Immunol. 1, 305–310. Curiel, T.J., Coukos, G., Zou, L., Alvarez, X., Cheng, P., Mottram, P., Evdemon-Hogan, M., Conejo-Garcia, J.R., Zhang, L., Burow, M., Zhu, Y., Wei, S., Kryczek, I., Daniel, B., Gordon, A., Myers, L., Lackner, A., Disis, M.L., Knutson, K.L., Chen, L., Zou, W., 2004. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 10, 942–949. Goddard, R.V., Prentice, A.G., Copplestone, J.A., Kaminski, E.R., 2001. Generation in vitro of B-cell chronic lymphocytic leukaemia-proliferative and specific HLA class-II-restricted cytotoxic T-cell responses using autologous dendritic cells pulsed with tumour cell lysate. Clin. Exp. Immunol. 126, 16–28. Goddard, R.V., Prentice, A.G., Copplestone, J.A., Kaminski, E.R., 2003. In vitro dendritic cell-induced T cell responses to B cell chronic lymphocytic leukaemia enhanced by IL-15 and dendritic cell-B-CLL electrofusion hybrids. Clin. Exp. Immunol. 131, 82–89. Hus, I., Rolinski, J., Tabarkiewicz, J., Wojas, K., BojarskaJunak, A., Greiner, J., Giannopoulos, K., Dmoszynska, A., Schmitt, M., 2005. Allogeneic dendritic cells pulsed with tumor lysates or apoptotic bodies as immunotherapy for patients with early-stage B-cell chronic lymphocytic leukemia. Leukemia 19, 1621–1627. Mohty, M., Jarrossay, D., Lafage-Pochitaloff, M., Zandotti, C., Briere, F., de Lamballeri, X.N., Isnardon, D., Sainty, D., Olive, D., Gaugler, B., 2001. Circulating blood dendritic cells

from myeloid leukemia patients display quantitative and cytogenetic abnormalities as well as functional impairment. Blood 98, 3750–3756. Mohty, M., Vialle-Castellano, A., Nunes, J.A., Isnardon, D., Olive, D., Gaugler, B., 2003. IFN-alpha skews monocyte differentiation into Toll-like receptor 7-expressing dendritic cells with potent functional activities. J. Immunol. 171, 3385–3393. Orsini, E., Guarini, A., Chiaretti, S., Mauro, F.R., Foa, R., 2003. The circulating dendritic cell compartment in patients with chronic lymphocytic leukemia is severely defective and unable to stimulate an effective T-cell response. Cancer Res. 63, 4497–4506. Rissoan, M.C., Soumelis, V., Kadowaki, N., Grouard, G., Briere, F., de Waal Malefyt, R., Liu, Y.J., 1999. Reciprocal control of T helper cell and dendritic cell differentiation. Science 283, 1183–1186. Scrivener, S., Kaminski, E.R., Demaine, A., Prentice, A.G., 2001. Analysis of the expression of critical activation/ interaction markers on peripheral blood T cells in B-cell chronic lymphocytic leukaemia: evidence of immune dysregulation. Br. J. Haematol. 112, 959–964. Zou, W., 2005. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat. Rev. Cancer 5, 263–274. Zou, W., Machelon, V., Coulomb-L’Hermin, A., Borvak, J., Nome, F., Isaeva, T., Wei, S., Krzysiek, R., DurandGasselin, I., Gordon, A., Pustilnik, T., Curiel, D.T., Galanaud, P., Capron, F., Emilie, D., Curiel, T.J., 2001. Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells. Nat. Med. 7, 1339–1346.