Impact of plasmacytoid dendritic cells on outcome after reduced-intensity conditioning allogeneic stem cell transplantation

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1 (2005) 19, 1 6 & 2005 Nature Publishing Group All rights reserved /05 $ Impact of plasmacytoid dendritic cells on outcome after reduced-intensity conditioning allogeneic stem cell transplantation M Mohty 1,2,3,7, D Blaise 1,2,4,7, C Faucher 1,2, V-J Bardou 5, J-A Gastaut 2,4, P Viens 4,6,7, D Olive 3,7 and B Gaugler 3,7 1 Unité de Transplantation et de Thérapie Cellulaire (UTTC), Institut Paoli-Calmettes, Marseille, France; 2 Département d Hématologie, Institut Paoli-Calmettes, Marseille, France; 3 Laboratoire d Immunologie des Tumeurs, Institut Paoli-Calmettes, Marseille, France; 4 Université de la Méditerranée, Marseille, France; 5 Unité de Biostatistiques, Institut Paoli-Calmettes, Marseille, France; 6 Département d Oncologie Médicale, Institut Paoli-Calmettes, Marseille, France; and 7 INSERM UMR 599, Marseille, France The reconstitution of the plasmacytoid dendritic cells (PDCs) compartment might influence outcome after allogeneic stem cell transplantation (allo-sct). Thus, we investigated the impact of blood PDCs measured at the third month after reduced-intensity conditioning (RIC) in 54 patients who received an HLA-identical sibling allo-sct. The absence of grade II IV acute graft-versus-host-disease (GVHD) was associated with an improved PDC count at 3 months after RIC-allo- SCT (P ¼ 0.003; OR ¼ 6.4; 95% CI, ). The CD34 þ stem cell dose and other lymphoid subsets infused with the allograft did not affect PDC recovery. Although PDC count could not predict death from progression or relapse, patients with a high PDC recovery profile had an improved overall survival (OS; P ¼ 0.03), in contrast to patients with a low PDC recovery profile who had an increased incidence of nonrelapse mortality (GVHD, infections) (P ¼ 0.03). The overall incidence of late infections (viral, fungal and bacterial) was significantly higher in the low PDC recovery group as compared to the high PDC recovery group (59 vs 19%; P ¼ 0.002). In a multivariate analysis, only a high PDC count was significantly predictive of a decreased risk of death (P ¼ 0.04; RR ¼ 0.34; 95% CI, ). Monitoring of PDCs at 3 months after RIC-allo-SCT may be a useful indicator predictor of long-term outcome. (2005) 19, 1 6. doi: /sj.leu Published online 4 November 2004 Keywords: plasmacytoid dendritic cell; reduced-intensity conditioning; allogeneic transplantation; innate immunity; infection Introduction In an attempt to decrease transplant-related toxicity, reducedintensity conditioning regimens (RIC) are increasingly being used prior to allogeneic stem cell transplantation (allo-sct). RIC-allo-SCT has been shown to be able to mediate a powerful graft-versus-tumor effect in patients with hematological and nonhematological malignancies. 1 3 The rapidly increasing use of RIC-allo-SCT emphasizes the need for clinical research aimed at identifying variables predictive of RIC-allo-SCT outcome. Dendritic cells (DCs) are crucial in the induction of immune responses. Among DC subsets, the reconstitution of the natural type I-interferon-producing plasmacytoid DCs (PDC) has been proposed to play a major role in establishing immune competence, given the capacity of PDCs to efficiently expand either specific cytotoxic T-lymphocytes 4 or to promote regulatory T cells, contributing to an impaired immune response. 5,6 Correspondence: M Mohty, MD, PhD, Unité de Transplantation et de Thérapie Cellulaire (UTTC), Institut Paoli-Calmettes, 232 Bd. Ste Marguerite, Marseille Cedex 09, France; Fax: þ ; mohtym@marseille.fnclcc.fr Received 10 August 2004; accepted 14 September 2004; Published online 4 November 2004 Therefore, we investigated the impact of circulating PDCs measured at the third month after RIC-allo-SCT, in 54 patients who received an RIC-allo-SCT from an HLA-identical sibling, in order to determine whether this could provide a convenient indicator for long-term outcome. Patients and methods Study design In all, 54 consecutive patients treated with RIC-allo-SCT in different trials in a single center (Institut Paoli-Calmettes) with a minimal follow-up of 3 months were included in this analysis. Written informed consent was obtained from each patient and donor. The studies were approved by the local ethical committee and performed according to institutional guidelines. The eligibility criteria for RIC-allo-SCT have been detailed elsewhere. 2,7 Patients, donors and grafts characteristics are summarized in Table 1. Transplant procedures The RIC regimen included either fludarabine, oral busulfan and various doses of antithymocyte globulin (thymoglobuline; SangStat, Lyon, France), 2 or fludarabine and low-dose total body irradiation. 8 Supportive care has been previously reported and was similar during the whole study period. 7 Graft-versushost-disease (GVHD) prophylaxis was carried out with cyclosporin A (CSA) alone or with CSA and mycophenolate mofetil. 8,9 The majority of patients (87%) received a peripheral blood stem cell allograft mobilized with G-CSF (10 mg/kg/day) for 5 days. Immunophenotyping of the allograft cellular content was performed using standard techniques. PDCs analysis PDCs analysis was performed for all 54 patients at a median of 92 days after allo-sct (range, ). Blood DCs were identified by three-color staining performed on PBMCs using the following monoclonal antibodies: ZM3.8-PC5 (mab against ILT3, an immunoglobulin-like transcript recently used to isolate DCs from the blood, mouse IgG1), 10 BU15-phycoerythrin (mab against CD11c) and fluorescein isothiocyanate-labeled mabs against lineage markers CD3, CD14, CD16, CD19 and CD56 (Beckman-Coulter, Marseille, France). Cells that did not label with these lineage markers were designated as lineage negative (lin ). PDCs were defined with the phenotype of lin /CD11c / ILT3 þ This reproducible technique has been routinely used in our laboratory for more than 5 years Cells were

2 2 Table 1 Demographic and graft characteristics of the low vs high PDC recovery groups at 3 months after transplantation Characteristic Low PDC group (PDCo0.725/ml), n ¼ 27 (%) High PDC group (PDCX 0.725/ ml), n ¼ 27 (%) P-value Patient age (years), 47 (18 61) 46 (33 63) NS median (range) Donor age (years), 40 (22 77) 48 (31 76) NS median (range) Female donor 16 (59) 13 (48) NS CMV serologic 4 (15) 3 (11) NS status, seronegative pair ABO mismatch 8 (30) 8 (30) NS Diagnosis Myeloid 9 (33) 11 (41) malignancy a Lymphoid 11 (41) 10 (37) NS malignancy b Nonhematological malignancy c 7 (26) 6 (22) Disease status, advanced disease 24 (89) 20 (74) NS Graft source Peripheral blood 25 (93) 22 (82) NS stem cells Bone marrow 2 (7) 5 (18) Graft composition ( 10 6 /kg recipient body weight), median (range) CD34+ stem cells 5.6 ( ) 5.3 ( ) NS CD3+ T cells 343 (14 618) 306 (15 642) NS CD4+ T cells 197 (5 431) 198 (8 453) NS CD8+ T cells 102 (6 407) 96 (6 255) NS CD19+ B cells 61 (2 167) 74 (5 183) NS CD56+ NK cells 36 (1 103) 32 (2 96) NS Conditioning regimen ATG-based RIC 21 (78) 21 (78) Without ATG 6 (22) 6 (22) NS a Number of PDCs / µl b IFN-alpha secretion pg/ml Median 0.21 Low PDC group High P=0.002 Median 2.65 GVHD prophylaxis CSA alone 20 (74) 16 (59) CSA and MMF 7 (26) 11 (41) NS a In all, 15 acute myeloid leukemia, 3 chronic myeloid leukemia and 2 myelodysplastic syndromes. b In all, 6 non-hodgkin lymphomas, 14 multiple myelomas and 1 chronic lymphocytic leukemia. c Nonhematological metastatic malignancies included 2 renal, 7 breast, 1 ovarian, 2 melanoma and 1 other. 3 CMV, cytomegalovirus; ATG, antithymocyte globulin; RIC, reducedintensity conditioning; CSA, cyclosporin A; MMF, mycophenolate mofetil; NK, natural killer; GVHD, graft-versus-host disease; NS, not significant. 0 II-IV 0-I Acute GVHD Figure 1 (a) Distribution of PDCs in the low vs high recovery groups determined at the third month after RIC-allo-SCT. The limit between the low and high recovery groups was defined as the median of PDCs (0.725 cells/ml) quantified at the third month after RIC-allo-SCT in the whole study population. (b) IFN-a secretion by PDCs after microbial stimulation according to agvhd grade. A total of 38 patients from this series could be tested after the onset of agvhd (around day 60 post allo-sct; n ¼ 14 in the grade II IV group and n ¼ 24 in the grade 0 I group). Results represented as means and s.e.m. analyzed on a FACSCalibur cytometer using CellQuest software (Becton-Dickinson, San Jose, CA, USA). Other cell subsets frequencies (total white blood cells (WBCs), neutrophils, lymphocytes and monocytes) were obtained for all patients from a standard automated counter. The IFN-a secretion assay after viral stimulation was performed as described previously. 17,19 Briefly, 10 5 cells were plated in triplicate in 96- well round-bottomed plates and stimulated with herpes simplex virus (kind gift of Dr C Zandotti, Hôpital La Timone, Marseille, France) for 48 h. Supernatants were tested for their IFN-a content by ELISA (Bender MedSystems, Vienna, Austria). Clinical outcomes and statistical methods Acute and chronic GVHD (agvhd, cgvhd) were evaluated according to standard criteria. 20 Upon diagnosis of grade II IV agvhd, all patients were primarily treated with CSA and methylprednisolone (2 mg/kg/day). Data were computed using s.e.m. software (SILEX, Mirefleurs, France). The Mann Whitney test was used for comparison of continuous variables. Categorical variables were compared using the w 2 test. The probability of developing agvhd was depicted by calculating the cumulative incidence. Cumulative incidence estimates were

3 Table 2 Transplant-related events and outcome of the low vs high PDC recovery groups at 3 months after transplantation 3 Low PDC group (PDCo0.725/ml), n ¼ 27 (%) High PDC group (PDCX0.725/ml), n ¼ 27 (%) P-value Median time (day, range) to ANC 4500/ml 16 (0 21) 16 (11 22) NS Median time (day, range) to platelet /ml 12 (0 42) 10 (0 24) NS Agvhd Grade 0 I 11 (40) 22 (81) Grade II 8 (30) 4 (15) Grade III IV 8 (30) 1 (4) Positive CMV antigenemia 12 (44) 10 (37) NS Blood cell counts at 3 months (/ml, median, range) Total WBCs 4000 ( ) 3900 ( ) NS Neutrophils 2811 ( ) 2546 ( ) NS Lymphocytes 486 ( ) 828 ( ) Monocytes 336 ( ) 480 ( ) Plasmacytoid dendritic cells 0.21 ( ) 2.65 ( ) o10 4 Outcome No cgvhd 7 (26) 6 (22) NS Extensive/limited cgvhd 14/6 (74) 12 / 9 (78) Death from disease progression or relapse 6 (22) 4 (15) NS Median follow-up (days) of surviving patients (range) 374 ( ) 336 ( ) NS CMV, cytomegalovirus; GVHD, graft-versus-host disease; ANC, absolute neutrophil count; NS, not significant. Non-relapse mortality (%) Low PDC recovery group P=0.03 High PDC recovery group Months after transplantation Figure 2 Nonrelapse mortality according to the low vs high PDCs recovery groups determined at the third month after RIC-allo- SCT. Dashed lines, low PDC recovery group; solid lines, high PDC recovery group. also used to measure the probability of relapse or progression and transplant-related mortality. Probabilities of overall and progression-free survival (OS, PFS) were estimated from the time of transplantation using the Kaplan Meier product-limit estimates. Differences between groups were tested using the logrank test. A multiple logistic regression with backward stepwise model selection was used to depict predictive factors for PDCs recovery at 3 months after allo-sct. The association of time to death with the PDC count and other relevant variables was evaluated in a multivariate analysis with the use of Cox s proportional-hazard regression model. Results and discussion The median absolute count of PDCs measured at 3 months after RIC-allo-SCT was 0.725/ml (range, ). Thus, for the purpose of allocating patients to low vs a high PDC recovery group, the median value of 0.725/ml was used (Figure 1a). Baseline demographic and graft characteristics for both groups are shown in Table 1. Overall, the cumulative incidence of grade II IV agvhd (median onset, 34 days) was 39% (95% CI, 26 52%), with this being significantly higher in the low PDC recovery group at 3 months (59 vs 19%; P ¼ 0.005; Table 2). In a multiple logistic regression analysis including all relevant parameters (demographic and graft characteristics, RIC regimens, CMV infections and agvhd), only the absence of clinically significant grade II IV agvhd was associated with an improved PDC recovery at 3 months (P ¼ 0.003; OR ¼ 6.4; 95% CI, ). Being the major type I IFN-secreting cells, we also investigated whether PDCs recovered after allo-sct are functional in response to viral stimulation. Patients experiencing grade 0 I agvhd could secrete significantly higher amounts of IFN-a as compared to patients with grade II IV agvhd (mean, 91 vs 0 pg/ml respectively; P ¼ 0.002; Figure 1b), likely highlighting the deleterious impact of corticosteroids therapy on PDC function as described previously. 21,22 As previously shown in the myeloablative allo-sct setting, the CD34 þ stem cell dose and other lymphoid subsets infused with the allograft did not correlate with PDC recovery after RICallo-SCT. 23 Patients with a low PDC recovery had also significantly decreased lymphocyte, monocyte, but not neutrophil counts in comparison with the high PDC recovery group

4 4 Figure 3 (a) OS in the whole study population. (b) PFS according to the low vs high PDCs recovery groups determined at the third month after RIC-allo-SCT. (c) OS according to the low vs high PDCs recovery groups determined at the third month after RIC-allo-SCT. Dashed lines, low PDC recovery group; solid lines, high PDC recovery group. (P ¼ and 0.025, respectively; Table 2). Patients from the low and high PDC recovery groups had a comparable rate of cgvhd (P ¼ NS). Deaths from progression or relapse were comparable between both groups (P ¼ NS). However, nonrelapse mortality was significantly higher in the low PDC group, since six patients in the low PDC recovery group died of refractory cgvhd (n ¼ 3) or infection (n ¼ 3) as compared to none of the patients in the high PDC recovery group (P ¼ 0.03; Figure 2). Interestingly, the overall incidence of late infections (viral, fungal and bacterial) was significantly higher in the low PDC recovery group as compared to the high PDC recovery group (59 vs 19%; P ¼ 0.002), further illustrating the importance of PDCs in anti-infectious immune responses. 24 With a median follow-up of 11 (range, ) months for the 37 surviving patients (23 complete remissions, six partial remissions, two stable and six progressive diseases), the Kaplan Meier estimate of OS at 3 years is 51% (95% CI, 32 64%; Figure 3a). PFS was not significantly different between the low and high PDC recovery groups (P ¼ NS; Figure 3b). However, given the significantly increased nonrelapse mortality in the low PDC recovery group, patients from the high PDC recovery group had a significantly better OS (P ¼ 0.03; Figure 3c). After controlling for demographic, graft and transplant characteristics, agvhd, monocytes and lymphocytes counts, in the multivariate analysis, only a high PDC count measured at 3 months after allo-sct was significantly predictive of an improved OS and a decreased risk of death (P ¼ 0.04; RR ¼ 0.34; 95% CI, ). Recently, an ever-growing body of data highlighted the role of PDCs after allo-sct or solid organ transplantation. 21,22,25 28 The results from our study suggest that quantification of PDCs at 3 months after RIC-allo-SCT (widely used date for allo-sct patient assessment) 29 may be a simple but major indicator for long-term outcome, especially transplant-related mortality and OS. Interestingly, in patients receiving a myeloablative standard allo- SCT, Reddy et al 27 could show recently that a low DC recovery (including PDCs) at the time of engraftment is associated with a worsened early outcome. However, the latter study could not determine whether this is clinically relevant for RIC-allo-SCT patients. 27 In our study, the PDC count did not correlate with the risk of disease progression or relapse. This might be explained by the predominance in our series of high-risk patients with a high tumor burden (82%), a group where complex PDC tumor interactions 30 might preclude exact evaluation of the impact of one subset of DCs on antitumor immunity. In addition to quantification, functional studies are warranted to decipher the role of PDCs in antitumor immunity that is still under considerable discussion. 17,30,31 Nonetheless, although blood PDCs might not completely reflect the status of PDCs in other tissues, the increased mortality rate from infections and refractory GVHD in patients with low PDC recovery further establishes the crucial role of PDCs for both efficient immune defenses, 4 and maintenance of tolerance, 5,6,32,33 that would temper the severity of cgvhd. In this context, another determinant for GVHD development and severity might be PDC chimerism. 34 However, we and others have previously shown that selectively sorted circulating PDCs recovered after RIC-allo-SCT are of exclusive donor origin. 14,35 Although the group of patients analyzed in this study was rather heterogeneous as for diagnoses and transplant characteristics, precluding establishing the definitive role and impact of the PDC subset after allogeneic transplantation, one must admit that the role and impact of rare immune effector cells would tend to be more evident in truly RIC and less toxic regimens. Prospective studies focussing on PDCs, but also other DC subsets (myeloid CD11c þ and CD16 þ DC subsets) are still needed in homogeneous groups of patients. Nonetheless, from the results of this study, we can reasonably envision that monitoring of PDCs may be useful for patients management (closer surveillance, infection prophylaxis, etc), and may have a significant impact on the probability of a favorable outcome in the context of RIC-allo-SCT.

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