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• W2056823393 abstract "NK cells play an important role in hematopoietic stem cell transplantation (HCT) and in cross talk with dendritic cells (DCs) to induce primary T cell response against infection. Therefore, we hypothesized that blood DCs should augment NK cell function and reduce the risk of leukemia relapse after HCT. To test this hypothesis, we conducted laboratory and clinical studies in parallel. We found that although, phenotypically, NK cells could induce DC maturation and DCs could in turn increase activating marker expression on NK cells, paradoxically, both BDCA1+ myeloid DCs and BDCA4+ plasmacytoid DCs suppressed the function of NK cells. Patients who received an HLA-haploidentical graft containing a larger number of BDCA1+ DCs or BDCA4+ DCs had a higher risk of leukemia relapse and poorer survival. Further experiments indicated that the potent inhibition on NK cell cytokine production and cytotoxicity was mediated in part through the secretion of IL-10 by BDCA1+ DCs and IL-6 by BDCA4+ DCs. These results have significant implications for future HCT strategies. NK cells play an important role in hematopoietic stem cell transplantation (HCT) and in cross talk with dendritic cells (DCs) to induce primary T cell response against infection. Therefore, we hypothesized that blood DCs should augment NK cell function and reduce the risk of leukemia relapse after HCT. To test this hypothesis, we conducted laboratory and clinical studies in parallel. We found that although, phenotypically, NK cells could induce DC maturation and DCs could in turn increase activating marker expression on NK cells, paradoxically, both BDCA1+ myeloid DCs and BDCA4+ plasmacytoid DCs suppressed the function of NK cells. Patients who received an HLA-haploidentical graft containing a larger number of BDCA1+ DCs or BDCA4+ DCs had a higher risk of leukemia relapse and poorer survival. Further experiments indicated that the potent inhibition on NK cell cytokine production and cytotoxicity was mediated in part through the secretion of IL-10 by BDCA1+ DCs and IL-6 by BDCA4+ DCs. These results have significant implications for future HCT strategies. Natural killer (NK) cells play an important role in hematopoietic stem cell transplantation (HCT) for leukemia [1Velardi A. Ruggeri L. Mancusi A. et al.Clinical impact of natural killer cell reconstitution after allogeneic hematopoietic transplantation.Semin Immunopathol. 2008; 30: 489-503Crossref PubMed Scopus (24) Google Scholar, 2Lanier L.L. The role of natural killer cells in transplantation.Curr Opin Immunol. 1995; 7: 626-631Crossref PubMed Scopus (29) Google Scholar, 3Valiante N.M. Parham P. Natural killer cells, HLA class I molecules, and marrow transplantation.Biol Blood Marrow Transplant. 1997; 3: 229-235PubMed Google Scholar, 4Murphy W.J. Koh C.Y. Raziuddin A. Bennett M. Longo D.L. Immunobiology of natural killer cells and bone marrow transplantation: merging of basic and preclinical studies.Immunol Rev. 2001; 181: 279-289Crossref PubMed Scopus (64) Google Scholar]. It has been demonstrated that donor NK cells may promote engraftment, prevent graft-versus host disease (GVHD), control infections, and reduce the risk of leukemia relapse. We and others have demonstrated that NK cell alloreactivity is affected by many determinants, including donor-versus-recipient KIR ligand compatibility, KIR-HLA receptor-ligand mismatch, and the milieu of cytokines such as interleukin (IL)-12 and IL-18 [1Velardi A. Ruggeri L. Mancusi A. et al.Clinical impact of natural killer cell reconstitution after allogeneic hematopoietic transplantation.Semin Immunopathol. 2008; 30: 489-503Crossref PubMed Scopus (24) Google Scholar, 5Ruggeri L. Capanni M. Urbani E. et al.Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants.Science. 2002; 295: 2097-2100Crossref PubMed Scopus (2720) Google Scholar, 6Leung W. Iyengar R. Triplett B. et al.Comparison of killer Ig-like receptor genotyping and phenotyping for selection of allogeneic blood stem cell donors.J Immunol. 2005; 174: 6540-6545PubMed Google Scholar, 7Leung W. Iyengar R. Turner V. et al.Determinants of antileukemia effects of allogeneic NK cells.J Immunol. 2004; 172: 644-650PubMed Google Scholar]. Furthermore, the potency of NK activity appears to be augmented by a lymphopenic environment, especially T cell lymphopenia because T cell alloreactivity dominates that of NK cells [8Lowe E.J. Turner V. Handgretinger R. et al.T-cell alloreactivity dominates natural killer cell alloreactivity in minimally T-cell-depleted HLA-non-identical paediatric bone marrow transplantation.Br J Haematol. 2003; 123: 323-326Crossref PubMed Scopus (120) Google Scholar]. Besides the interaction with T cells, we began to investigate the interactions of human NK cells with dendritic cells (DCs) and examined their effects on patient outcomes after HCT. Blood DCs are not a single cell type but consist of several distinct subsets, including myeloid and lymphoid/plasmacytoid DC (pDC) that are derived from common myeloid and lymphoid progenitors, respectively [9MacDonald K.P. Munster D.J. Clark G.J. Dzionek A. Schmitz J. Hart D.N. Characterization of human blood dendritic cell subsets.Blood. 2002; 100: 4512-4520Crossref PubMed Scopus (629) Google Scholar, 10Ishikawa F. Niiro H. Iino T. et al.The developmental program of human dendritic cells is operated independently of conventional myeloid and lymphoid pathways.Blood. 2007; 110: 3591-3660Crossref PubMed Scopus (88) Google Scholar]. The interactions between DCs and NK cells have been well documented [11Cooper M.A. Fehniger T.A. Fuchs A. Colonna M. Caligiuri M.A. NK cell and DC interactions.Trends Immunol. 2004; 25: 47-52Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 12Fernandez N.C. Lozier A. Flament C. et al.Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo.Nat Med. 1999; 5: 405-411Crossref PubMed Scopus (925) Google Scholar, 13Marcenaro E. Dondero A. Moretta A. Multi-directional cross-regulation of NK cell function during innate immune responses.Transpl Immunol. 2006; 17: 16-19Crossref PubMed Scopus (38) Google Scholar, 14Walzer T. Dalod M. Robbins S.H. Zitvogel L. Vivier E. Natural-killer cells and dendritic cells: “l’union fait la force”.Blood. 2005; 106: 2252-2258Crossref PubMed Scopus (484) Google Scholar, 15Wilson J.L. Heffler L.C. Charo J. Scheynius A. Bejarano M.T. Ljunggren H.G. Targeting of human dendritic cells by autologous NK cells.J Immunol. 1999; 163: 6365-6370PubMed Google Scholar]. For instance, in infection, it is well known that DCs and NK cells crosstalk to induce primary T cell response [14Walzer T. Dalod M. Robbins S.H. Zitvogel L. Vivier E. Natural-killer cells and dendritic cells: “l’union fait la force”.Blood. 2005; 106: 2252-2258Crossref PubMed Scopus (484) Google Scholar, 16Della Chiesa M. Sivori S. Castriconi R. Marcenaro E. Moretta A. Pathogen-induced private conversations between natural killer and dendritic cells.Trends Microbiol. 2005; 13: 128-136Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar]. Both NK cells and DCs are part of the innate immune response system and are equipped with a large array of pattern recognition receptors (PRRs) that allow the recognition of different pathogens [17Medzhitov R. Recognition of microorganisms and activation of the immune response.Nature. 2007; 449: 819-826Crossref PubMed Scopus (2035) Google Scholar]. Toll-like receptors (TLRs) are PRRs that upon recognition of pathogen-associated molecular patterns (PAMPs) can induce triggering of innate immune response [18Aderem A. Ulevitch R.J. Toll-like receptors in the induction of the innate immune response.Nature. 2000; 406: 782-787Crossref PubMed Scopus (2632) Google Scholar]. When immature DCs (iDCs) are activated by TLR ligands, they differentiate into mature DCs (mDCs) that express high levels of MHC class I and MHC class II antigens as well as costimulatory molecules (CD80, CD86), which are required for stimulation of naïve T cells [19Andoniou C.E. van Dommelen S.L. Voigt V. et al.Interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity.Nat Immunol. 2005; 6: 1011-1019Crossref PubMed Scopus (232) Google Scholar, 20Reis e Sousa C. Toll-like receptors and dendritic cells: for whom the bug tolls.Semin Immunol. 2004; 16: 27-34Crossref PubMed Scopus (287) Google Scholar]. Because HCT setting is characterized by an inflammatory environment with abundant PAMPs derived from endogenous pathogens and host tissue damaged by the high-dose chemotherapy and radiation [21Taylor P.A. Ehrhardt M.J. Lees C.J. et al.TLR agonists regulate alloresponses and uncover a critical role for donor APCs in allogeneic bone marrow rejection.Blood. 2008; 112: 3508-3516Crossref PubMed Scopus (67) Google Scholar, 22Cooke K.R. Gerbitz A. Crawford J.M. et al.LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation.J Clin Invest. 2001; 107: 1581-1589Crossref PubMed Scopus (245) Google Scholar, 23Blazar B.R. Krieg A.M. Taylor P.A. Synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides are potent stimulators of antileukemia responses in naive and bone marrow transplant recipients.Blood. 2001; 98: 1217-1225Crossref PubMed Scopus (78) Google Scholar], we hypothesized that DCs should augment NK cell function and thereby reduce the risk of leukemia relapse after transplantation. To test this hypothesis, we conducted laboratory and clinical studies in parallel to examine the effects of blood DC subsets on NK cells in HCT setting. The following fluorochrome-labeled monoclonal antibodies (mAbs) against human antigens were obtained from BD Pharmigen (San Jose, CA): CD3-APC, CD8-PECy7, CD11c-APC, CD14-APCCy7, CD19-APCCy7, CD20-PE, CD45-FITC, CD69-FITC, CD80-FITC, CD81-PE, CD83-PECy5, CD123-PE, NKG2D-APC, IFN-γ-FITC, IFN-γ-PECy7, Lin markers cocktail-FITC, Phospho-Zap70 (Y319)/Syk (Y352)-PE, and Phospho-SLP-76 (pY128)-PE. Fluorochrome-labeled mAbs CD25-PE, CD9-FITC, and CD19-PE were obtained from DAKO (Carpinteria, California). Fluorochrome-labeled mAbs CD3-ECD, CD45-ECD, CD56-APC, CD86-PE, and HLA DR-ECD were obtained from Beckman Coulter (Fullerton, CA). Fluorochrome-labeled mAbs CD1c (BDCA1)-PE and CD304 (BDCA4)-APC were obtained from Miltenyi Biotec (Auburn, CA). A-Class CpG ODN 2216 5′-ggGGGAGCATGCTGgggggG-3′ as TLR9 ligand was provided by Hartwell Center for Bioinformatics and Biotechnology at St. Jude’s Children’s Research Hospital. Lipopolysaccharide (LPS, Sigma 0127:B8) was used as a TLR4 ligand. K562 leukemia cell lines were used as targets for NK cell natural cytotoxicity assays. Luciferase transduced neuroblastoma cell line (NB1691luc) was kindly provided by Dr. A. Davidoff (St. Jude’s Children’s Research Hospital) and was used as the target in a quantitative in vivo mouse model. NK cells were magnetically isolated from nonmobilizied apheresis products of healthy donor using the AutoMACS machine and NK cell isolation kit from Miltenyi Biotec following the manufacturer’s specifications. From each 1-2 × 108 peripheral blood mononuclear cell (PBMC) product, we typically obtained between 5 to 15 × 106 cells with a purity of always >95% CD56+CD3− cells. Donor HLA typing was not performed. BDCA1+ myeloid DCs and BDCA4+ pDCs were magnetically isolated using the AutoMACS machine and Human CD1c (BDCA1) dendritic cell isolation kit and CD304 (BDCA-4/Neuropilin-1) kits from Miltenyi Biotec following the manufacturer’s specifications. Starting with 2-8 × 109 PBMCs, between 0.5-5 × 106 cells were typically obtained after the isolation procedures ending with a purity always >95% for BDCA4+ cells and >90% for BDCA1+ cells. High levels of expression of TLR9 in purified BDCA4+ cell products and TLR4 in purified BDCA1+ products were confirmed by RTPCR. Freshly purified NK cells were cocultured with or without DC subsets in a cell ratio of 2:1 overnight with or without adding LPS 100 ng/mL or a-CpG 10 μg/mL. Cultures were performed in complete culture medium (RPMI 1640 supplemented with 10% of heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, 100 ng/mL streptomycin, and 2 mM/L-glutamine) in a humidified atmosphere of 5% CO2 and 95% air, with no addition of exogenous cytokines such as IL-2 or IL-15. DC maturation was assessed using CD80, CD83, and HLA-DR mAbs. CD69 and CD25 were used to assess NK cell activation status. RNA was isolated with RNAeasy (Qiagen 2001, Chatsworth, CA). DNA was removed by digestion with 5 U deoxyribonuclease I (Boehringer, Indianapolis, IN) for 30 minutes at 37°C. Reverse transcription (RT) was performed with oligo-dT primer (Omniscript, Qiagen 2004). The PCR reaction volume was 50 μL, containing 0.5 μM of each primer, 200 μM of each dNTP, and 2.5 units HotStarTaq (Qiagen 2005). The following sets of primers were used: TLR4 (F: 5′-CTGCAATGGATCAAGGACCA-3′, R: 5′-TTATCTGAAGGTGTTGCACATTCC-3′) and TLR9 (F: 5′-TGAAGACTTCAGGCCCAACTG-3′, R: 5′-TGCACGGTCACCAGGTTGT-3′). A GeneAmp PCR System 9700 (Perkin-Elmer/Applied Biosystems, Bedford, MA) was used with an initial denaturation step of 95°C for 15 minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds (annealing temperature), 72°C for 1 minute (extension temperature), and a final elongation step of 72°C for 7 minutes. Real-time quantitative PCR (RQ-PCR) was performed by using the ABI Prism 7700 Sequence Detector Systems (Applied Biosystems) and the SYBR Green I Dye assay chemistry, as suggested by the manufacturer. Briefly, all reactions were performed with 2 μL (80 ng) of cDNA, 12.5 μL of SYBR GREEN PCR master mix (Applied Biosystems), and 0.5 μM forward and reverse primers in a final reaction volume of 25 μL. A water control and melting curve analysis were always performed to confirm the specificity of the PCR. GAPDH was used as an internal control to normalize the difference in the amount of cDNA contained in each initial reaction. Reactions were incubated for 2 minutes at 50°C, denatured for 10 minutes at 95°C, and subjected to 40 2-step amplification cycles with annealing/extension at 60°C for 1 minute followed by denaturation at 95°C for 15 seconds. NK cell signaling after direct TLR ligand stimulation was determined by multiparameter intracellular phospho-proteins assay using flow cytometry. In brief, 1 × 106 unstimulated NK cells or NK cells stimulated with LPS or a-CpG were fixed after different time points (0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300 seconds, 15, 30 minutes, and 8 hours) by adding 4% formaldehyde directly into the culture medium to obtain a final concentration of 2% formaldehyde. NK cells were incubated in fixative for 10 minutes at ambient temperature and pelleted. They were then permeabilized by resuspending with vigorous vortexing in 500 μL of ice-cold MeOH per 106 and incubated at 4°C for 30 minutes. After that, cells were washed twice in staining medium (PBS containing 1% BSA) and then resuspended in staining medium at 0.5-1 × 106 cells per 100 μL. Fluorophore-specific mAbs were added and incubated for 15 to 30 minutes at ambient temperature. The cells were washed with 15 volumes of staining medium and pelleted. Finally, samples were resuspended in 100 μL of staining medium and analyzed. At the same time points, intracellular IFN-γ staining was analyzed with mouse antihuman-IFN-γ clone 4S.B3. Purified NK cells (2 × 105), with or without DC subsets (1 × 105), were cultured in 48-well plates in 0.4 mL of complete culture medium and incubated with or without 10 μg/mL CpG or 100 ng/mL LPS at 37°C and 5% CO2. After 15 h of incubation, culture supernatants were collected and analyzed for cytokine production by ELISA using the Bioplex Protein Array system (BioRad, Hercules, CA) according to the instructions of the manufacturer. Data analyses of all assays were performed with the Bio-Plex Manager software. Natural cytotoxicity of NK cells was assessed in a conventional 2-hour europium-TDA release assay (Perkin-Elmer Wallac, Turku, Finland) [24Bari R. Bell T. Leung W.H. et al.Significant functional heterogeneity among KIR2DL1 alleles and a pivotal role of arginine 245.Blood. 2009; 114: 5182-5190Crossref PubMed Scopus (84) Google Scholar]. The following formulas were used to calculate spontaneous and specific cytotoxicity:%Specificrelease=(Experimentalrelease−spontaneousrelease)/(Maximumrelease−spontaneousrelease)×100.%Spontaneousrelease=(Spontaneousrelease-background)/(Maximumrelease-background)×100. Purified NK cells were incubated at 37°C with various concentrations of recombinant human IL-6 or recombinant human IL-10 (R&D Systems, Minneapolis, MN). Cytotoxic function of NK cells was determined using a standard europium-release assay as described above after 48 hours of incubation. Six- to 8-week-old NOD-scid IL2Rgnull mice were sublethally irradiated with 200 cGy and 24 hours later they were injected intravenously with NB-1691luc 5 × 105 neuroblastoma cells. Intravenous NK cellular therapy began 7 days after the injection of tumor cells and was performed once a week for 3 weeks. In 3 independent experiments (15 mice per group), we compared untreated mice (control group) with mice receiving 1 × 106 NK cells stimulated overnight with 10 μg/mL a-CpG (NK + CpG group), or mice receiving 1 × 106 NK cells and 5 × 105 BDCA4 in coculture overnight with 10 μg/mL a-CpG (NK + CpG + BDCA4 group). Bioluminescence imaging was performed after the initiation of NK cell therapy on days 7, 14, and 21 after intraperitoneal (i.p.) injection of 200 μL of luciferin dissolved in phosphate-buffered saline (PBS) at a concentration of 15 mg/mL. Between 7 and 10 minutes after administration of substrate, animals were anesthetized using isofluorane and transferred to the Xenogen IVIS-200 Imaging system (Xenogen Corporation, Hopkinton, MA). Images were captured at varied exposures and analysis performed using Xenogen Living Image Software (version 2.50). For BLI plots, a rectangular region of interest (ROI) encompassing the entire thorax and abdomen was applied for each mouse and total flux (photons/second [p/s]) calculated in ventral and prone positions at different exposures (1, 10, 60, and 120 seconds). This value was scaled to a comparable background value (from a nontumor-bearing, luciferin-injected control mouse). All experiments were conducted following the guidelines of the Institutional Animal Care and Use Committees according to criteria outlined in the NIH Guide for Care and Use of Laboratory Animals. Patients received either total-body irradiation (TBI, 12Gy) with thiotepa (10 mg/kg) and cyclophosphamide (120 mg/kg), or a non-TBI regimen with fludarabine (150 mg/m2), thiotepa (10 mg/kg), and melphalan (140 mg/m2). Post-HCT, patients required only single agent GVHD prophylaxis with cyclosporine or mycophenolate, because all the grafts were depleted of CD3+ cells by immunomagnetic method using the CliniMACs system (Miltenyi). The number of BDCA1+ cells and BDCA4+ cells in the parental grafts after T cell depletion were measured by flow cytometry analysis. The risk of leukemia relapse and death after HCT was estimated and compared by using the Kaplan-Meier method and log-rank test among patients who were categorized in tertiles based on the number of donor BDCA1+ cells (<0.5, 0.5-1.0, and >1.0 × 108) or BDCA4+ cells (<1.4, 1.5-2.5, and >2.5 × 108) in the grafts. Other risk factor analyses included age, primary diagnosis, degree of HLA match, conditioning, GVHD prophylaxis, history of GVHD, and remission status. The standard risk category consisted of patients with acute myelegenous leukemia (AML), acute lymphobalstic leukemia (ALL), or non-Hodgkin leukemia (NHL) in first or second complete remission. The high-risk category included patients in third or subsequent remission, in relapse, or with myelodysplastic syndrome (MDS). The effects of blood DCs on the cytokine production and cytotoxicity of NK cells in vitro were evaluated by Wilcoxon rank sum tests. The dose-response suppressive effects of IL-6 and IL-10 on NK cell cytotoxicity were analyzed by trend test. The difference in total flux in bioluminescence was compared using repeated-measures analysis of variance (ANOVA). Highly purified CD56+3− NK cells were colocalized in our in vitro culture system with either BDCA1+ myeloid DCs or BDCA4+ pDCs for 24 hours in cell ratio of 2:1. We observed an increase in surface expression in both DC subsets of costimulatory molecules (CD80), maturation markers (CD83), and MHC Class II (HLA-DR); all of these changes suggested the blood DCs were undergoing maturation in the in vitro system. In turn, both DC subsets were able to activate NK cells phenotypically, as indicated by the induction of CD69 and CD25 expression on NK cells. On the basis of the phenotypic changes described above, we hypothesized that human blood DCs should augment the function of NK cells in the presence of TLR ligands in the HCT setting. To test this hypothesis, we examined the direct effect of TLR ligands on NK cells and the secondary effects through DCs. We found that both TLR4 and TLR9 were expressed on highly purified NK cells as determined by RT-PCR assay, but the level of mRNA did not change with ligand exposure as demonstrated by RQ-PCR analysis (mean delta, cycle threshold (CT) 7.9 ± 1.4 versus 7.7 ± 1.1 for TLR4, and 5.8 ± 1.7 versus 5.4 ± 1.4, respectively, all P > .48). Importantly, the TLRs were functional as demonstrated by positive ZAP70/SYK and SLP76 signaling and a rise in production of IFN-γ (Figure 1). Furthermore, TLR ligands increased the secretion of IFN-γ by NK cells (Figure 2A) and enhanced the cytotoxicity against K562 cells (Figure 2B). Surprisingly, both BDCA1+ myeloid DC and BDCA4+ pDC strongly suppressed the NK cell production of IFN-γ induced by TLR ligands (Figure 2A). In addition, BDCA4+ pDC were able to reduce the stimulation effect of a-CpG on NK cytotoxicity against K562 cells, whereas BDCA1+ myeloid DC could completely offset the effect of LPS (Figure 2B).Figure 2Effects of TLR ligands and blood DC subsets on IFN-γ secretion and natural cytotoxicity of NK cells. (A) IFN-γ secretion by unstimulated NK cells (NK), NK cells stimulated overnight with 100 ng/mL of LPS (NK + LPS) or 10 μg/mL of a-CpG (NK + CpG), and NK cells stimulated overnight with 1 of these two TLR ligands in the presence of BDCA1+ myeloid DCs (NK + LPS + BDCA1) or BDCA4+ pDCs (NK + CpG + BDCA4) in a starting 2:1 cell ratio (no change in cell number or ratio thereafter was observed overnight). Results are expressed as means ± SEM from 3 independent experiments. (B) Cytotoxicity against K562 cells (NK:K562 cell ratio were always 8:1) by unstimulated NK cells (NK), NK cells stimulated overnight with 100 ng/mL of LPS (NK + LPS) or 10 μg/mL of a-CpG (NK + CpG), and NK cells stimulated overnight with 1 of these two TLR ligands in the presence of BDCA1+ myeloid DCs (NK + LPS + BDCA1) or BDCA4+ pDCs (NK + CpG + BDCA4) in a NK:DC cell ratio of 2:1. After cocultured, NK cells were not further purified. Results are expressed as means ± SEM from 3 independent experiments. ∗P < .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then extended our investigation to an in vivo model to examine whether the modest suppressive effect of BDCA4+ pDC on TLR ligand stimulated NK cells in vitro might be clinically meaningful. We found that BDCA4+ pDC were indeed able to suppress the antitumor effect of CpG-activated NK cells (P < .05) (Figure 3). To examine the effects of DC subsets on patient outcomes, we studied 47 patients with AML (n = 17), ALL (n = 25), or NHL (n = 5) who received an HCT from a HLA-haploidentical parental donor. We measured the number of DC in the patient’s graft prospectively and categorized them into tertiles based on the DC content. We found that those patients who had in their graft a higher number of BDCA1+ myeloid DC (upper tertile) or BDCA4+ pDC (upper 2 tertiles) had poorer survival because of increased incidence in leukemia relapse (Figure 4). No other factors were found to be statistically significant, including age, primary diagnosis, risk group, degree of HLA matching, conditioning, GVHD prophylaxis, or history of GVHD (all P > .16). The number of BDCA1+ or BDCA4+ DCs in the graft has no effect on transplant-related mortality (P > .36). To investigate the mechanisms of the suppressive effects of blood DCs on NK cells activated by TLR ligands, we examined the cytokine profile of the in vitro cocultures. We found that in the presence of BDC1+ myeloid DC or BDC4+ pDC, there was a large amount of IL-10 (mean 600 pg/mL) or IL-6 (mean 2000 pg/mL), respectively (Figure 5A). Incubation of NK cells with recombinant IL-6 or IL-10 at these cytokine concentrations confirmed a dose-dependent suppressive effects on the cytotoxic function of NK cells (Figure 5B). In this study, we sought to prove that blood DCs augmented NK cell function and thus favored the clinical activity of NK cells against AML [5Ruggeri L. Capanni M. Urbani E. et al.Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants.Science. 2002; 295: 2097-2100Crossref PubMed Scopus (2720) Google Scholar] and ALL [7Leung W. Iyengar R. Turner V. et al.Determinants of antileukemia effects of allogeneic NK cells.J Immunol. 2004; 172: 644-650PubMed Google Scholar]. Our hypothesis was based on the large body of literatures supporting a critical role of DC and NK cell crosstalk in the control of infection [18Aderem A. Ulevitch R.J. Toll-like receptors in the induction of the innate immune response.Nature. 2000; 406: 782-787Crossref PubMed Scopus (2632) Google Scholar, 25Andrews D.M. Scalzo A.A. Yokoyama W.M. Smyth M.J. Degli-Esposti M.A. Functional interactions between dendritic cells and NK cells during viral infection.Nat Immunol. 2003; 4: 175-181Crossref PubMed Scopus (312) Google Scholar, 26Guan H. Moretto M. Bzik D.J. Gigley J. Khan I.A. NK cells enhance dendritic cell response against parasite antigens via NKG2D pathway.J Immunol. 2007; 179: 590-596PubMed Google Scholar, 27Gerosa F. Baldani-Guerra B. Nisii C. Marchesini V. Carra G. Trinchieri G. Reciprocal activating interaction between natural killer cells and dendritic cells.J Exp Med. 2002; 195: 327-333Crossref PubMed Scopus (880) Google Scholar]. Surprisingly, we found that although, phenotypically, NK cells could induce DC maturation and DCs could in turn increase the expression of activating markers in NK cells as described by other investigators, paradoxically, both blood BDCA1+ myeloid DC and BDCA4+ pDC suppressed the function of NK cells in both in vitro assays and in vivo mouse models when TLR ligands were used to mimic the microbe-associated inflammatory environment in allogeneic HCT [28Penack O, Holler E, van den Brink MR. Graft-versus-host disease: regulation by microbe-associated molecules and innate immune receptors. Blood. 115:1865-1872.Google Scholar]. The clinical relevance of these finding is underscored by our observation that patients who received an HLA-haploidentical graft containing large number of BDCA1+ myeloid DC and BDCA4+ pDC had a high risk of leukemia relapse. Detailed biologic studies reveal that DC were a potent inhibitor of both NK cell cytotoxicity and cytokine production and were mechanistically mediated in part through IL-10 or IL-6. These findings have significant implications for future HCT strategies. NK cell function is tightly regulated by a balance of signals from inhibitory and activating receptors. The “missing-self” hypothesis suggests that NK cell specificity is determined by the interaction between NK cell inhibitory receptors and target cell MHC class I expression [29Karre K. Natural killer cell recognition of missing self.Nat Immunol. 2008; 9: 477-480Crossref PubMed Scopus (201) Google Scholar]. Even in the absence of a target cell major histocompability complex (MHC), however, most activating receptor-ligand interactions are insufficient to induce cytotoxicity by resting peripheral-blood NK cells [30Bryceson Y.T. March M.E. Ljunggren H.G. Long E.O. Activation, coactivation, and costimulation of resting human natural killer cells.Immunol Rev. 2006; 214: 73-91Crossref PubMed Scopus (455) Google Scholar]. Except for CD16, preactivation of NK cells is required to generate sufficient signal for cytolytic granule polarization and degranulation [31Bryceson Y.T. March M.E. Barber D.F. Ljunggren H.G. Long E.O. Cytolytic granule polarization and degranulation controlled by different receptors in resting NK cells.J Exp Med. 2005; 202: 1001-1012Crossref PubMed Scopus (366) Google Scholar, 32Bryceson Y.T. March M.E. Ljunggren H.G. Long E.O. Synergy among receptors on resting NK cells for the activation of natural cytotoxicity and cytokine secretion.Blood. 2006; 107: 159-166Crossref PubMed Scopus (585) Google Scholar]. NK cell may be preactivated by many non-antibody-directed cell-mediated cytotoxicity (ADCC) factors such as cytokines or pathogen-derived substances [30Bryceson Y.T. March M.E. Ljunggren H.G. Long E.O. Activation, coactivation, and costimulation of resting human natural killer cells.Immunol Rev. 2006; 214: 73-91Crossref PubMed Scopus (455) Google Scholar]. TLR ligands are pathogen-derived substances that can activate NK cell indirectly through accessory cells [33Hornung V. Rothenfusser S. Britsch S. et al.Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides.J Immunol. 2002; 168: 4531-4537PubMed Google Scholar, 3" @default.
• W2056823393 created "2016-06-24" @default.
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• W2056823393 date "2011-05-01" @default.
• W2056823393 modified "2023-09-24" @default.
• W2056823393 title "Blood Dendritic Cells Suppress NK Cell Function and Increase the Risk of Leukemia Relapse after Hematopoietic Cell Transplantation" @default.
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