Impaired Interferon-Alpha Production by Plasmacytoid Dendritic Cells after Cord Blood Transplantation in Children: Implication for Post-transplantation.

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Impaired Interferon-Alpha Production by Plasmacytoid Dendritic Cells after Cord Blood Transplantation in Children: Implication for Post-transplantation Toll-Like Receptor Ligand–Based Immunotherapy Emily Charrier, Paulo Cordeiro, Rose-Marie Brito, Michaël Harnois, Samira Mezziani, Sabine Herblot, Françoise Le Deist, Michel Duval Biology of Blood and Marrow Transplantation Volume 20, Issue 10, Pages 1501-1507 (October 2014) DOI: 10.1016/j.bbmt.2014.06.007 Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Plasmacytoid dendritic cell counts after cord blood and bone marrow transplantation. Lin− CD123high HLA-DR+ plasmacytoid dendritic cell (pDC) counts from healthy volunteers (HV, gray squares, n = 13), bone marrow- (BMT, white triangles, n = 17) and cord blood- transplant recipients (CBT, black circles, n = 26) patients at 1, 2, 3, 6, 9, and 12 months after transplantation. (A) Related and unrelated transplant recipients. (B) Unrelated transplant recipients only. Each symbol represents 1 patient. Horizontal bars represent the means. A 2-sided Mann-Whitney test was used to compare CBT patients with BMT patients (P values are indicated at the top of each time-point) and to healthy volunteers (*P < .05; **P < .01; ***P < .001). Biology of Blood and Marrow Transplantation 2014 20, 1501-1507DOI: (10.1016/j.bbmt.2014.06.007) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 In vitro differentiation of functional pDCs from CB and BM-derived CD34+ cells. CD34+ cells were purified from either CB or BM samples and cultured in the presence of TPO, SCF, and Flt3-L for 1 week, followed by 1 to 2 weeks of culture in the presence of Flt3-L, TPO, and interleukin-7. (A) Total cell expansion of CB and BM-CD34+ cells is represented (n = 4 for BM samples and n = 10 for CB samples). (B) Human pDCs obtained after 20 days of CD34+ cell culture are identified as CD123hiHLA-DR+BDCA2+ cells by flow cytometry. (C) Ten thousand in vitro differentiated human pDCs were sorted by flow cytometry and stimulated overnight by CpG ODN 2216 and IFN-α was measured in the supernatant by ELISA. Data are shown at the mean ± SEM of 5 independent experiments. (D) In vitro differentiated human pDCs were stimulated with CpG ODN 2216. The upregulation of activation markers (CD80, CD86, CD40, CD54) was assessed by flow cytometry. Data are representative of 3 independent experiments. Biology of Blood and Marrow Transplantation 2014 20, 1501-1507DOI: (10.1016/j.bbmt.2014.06.007) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Functional analysis of plasmacytoid dendritic cells after cord blood and bone marrow transplantation. (A) Expression of costimulatory molecules after TLR-9 stimulation by CpG 2216 was assessed by flow cytometry in samples from healthy volunteers (HV, gray bars, n = 9), and recipients of BMT (white bars, n = 12) or CBT (black bars, n = 12). The increase in expression of costimulatory molecules (CD40, CD54, CD80, and CD86) is expressed as Δ mean fluorescence intensity (Δ MFI = MFI of stimulated cells – MFI of unstimulated cells/MFI of unstimulated cells). Results are shown as mean ± SEM. Significant P values are reported as: *P < .05; **P < .01; ***P < .001. (B) Interferon-alpha (IFN-α) production after overnight toll-like receptor (TLR)-9 stimulation by CpG ODN 2216 was measured in peripheral blood mononuclear cells of healthy volunteers (HV, gray bars, n = 8), and recipients of bone marrow (BMT, white bars, n = 12) or cord blood transplantation (CBT, black bars, n = 15). IFN-α production was measured in the supernatants by ELISA. Results are expressed as IFN-α production by stimulated cells - IFN-α production by unstimulated cells and normalized for 104 pDCs. Results are shown as mean ± SEM. A 2-sided Mann-Whitney test was used to compare CBT patients with BMT patients (P values are indicated at the top of each time-point) and healthy volunteers (*P < .05; **P < .01; ***P < .001). Biology of Blood and Marrow Transplantation 2014 20, 1501-1507DOI: (10.1016/j.bbmt.2014.06.007) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions