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Circulating blood dendritic cells from myeloid leukemia patients display quantitative and cytogenetic abnormalities as well as functional impairment by.

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Presentation on theme: "Circulating blood dendritic cells from myeloid leukemia patients display quantitative and cytogenetic abnormalities as well as functional impairment by."— Presentation transcript:

1 Circulating blood dendritic cells from myeloid leukemia patients display quantitative and cytogenetic abnormalities as well as functional impairment by Mohamad Mohty, David Jarrossay, Marina Lafage-Pochitaloff, Christine Zandotti, Francine Brière, Xavier-Nicolas de Lamballeri, Daniel Isnardon, Danielle Sainty, Daniel Olive, and Béatrice Gaugler Blood Volume 98(13): December 15, 2001 ©2001 by American Society of Hematology

2 Detection of circulating DC subsets in the peripheral blood
Detection of circulating DC subsets in the peripheral blood.PBMCs isolated from healthy volunteers or AML patients were analyzed by flow cytometry after 3-color staining with a combination of FITC-labeled mAbs against lineage markers (CD14, CD16, CD19, and ... Detection of circulating DC subsets in the peripheral blood.PBMCs isolated from healthy volunteers or AML patients were analyzed by flow cytometry after 3-color staining with a combination of FITC-labeled mAbs against lineage markers (CD14, CD16, CD19, and CD56), PE-labeled anti-CD11c, and PC5-labeled ILT3. Two distinct populations of lin−/ILT3+ cells were observed with respect to the expression of CD11c, with the phenotypes of lin−/CD11c+/ILT3+ (MDCs) and lin−/CD11c−/ILT3+ (PDCs). Examples of expansion of the MDC subset for patient UPN109 or the PDC subset for patient UPN223 are shown. Mohamad Mohty et al. Blood 2001;98: ©2001 by American Society of Hematology

3 Immunophenotype of freshly isolated MDCs, PDCs, and leukemic blasts and expression of pre-Tα by leukemic PDCs.(A) Leukemic MDCs and PDCs were sorted as lin−/CD11c+/ILT3+ (MDCs) and lin−/CD11c−/ILT3+ (PDCs) and compared with blasts obtained from the same pat... Immunophenotype of freshly isolated MDCs, PDCs, and leukemic blasts and expression of pre-Tα by leukemic PDCs.(A) Leukemic MDCs and PDCs were sorted as lin−/CD11c+/ILT3+ (MDCs) and lin−/CD11c−/ILT3+ (PDCs) and compared with blasts obtained from the same patient for the expression of a number of lymphoid, myeloid, costimulatory, and cytokine receptor markers. Open histograms represent cells stained with isotype-matched control mAbs. Data shown were obtained from patient UPN243 and are representative of more than 5 patients with or without DC expansion. (B) Freshly isolated normal and leukemic MDCs and PDCs were analyzed for expression of mRNA for pre-Tα. Data shown are representative of 3 patients. Mohamad Mohty et al. Blood 2001;98: ©2001 by American Society of Hematology

4 Morphologic and confocal microscopy analysis of MDCs and PDCs isolated from AML patients.CD11c+ MDCs were isolated from patient UPN71 and cultured in vitro for 3 days with GM-CSF, IL-4, and CD40L. Morphologic and confocal microscopy analysis of MDCs and PDCs isolated from AML patients.CD11c+ MDCs were isolated from patient UPN71 and cultured in vitro for 3 days with GM-CSF, IL-4, and CD40L. (A) They display dendritic morphology as shown by interferential contrast transmission microscopy (×100). Three-color immunofluorescence staining was performed. (B) HLA-DR expression is shown in red, (C) CD83 in blue, and (D) DC-LAMP in green. (E) Giemsa staining of freshly isolated PDCs from patient UPN109. Original magnification × 1000. Mohamad Mohty et al. Blood 2001;98: ©2001 by American Society of Hematology

5 Leukemic status of MDCs and PDCs isolated from the blood of AML patients.MDCs from patient UPN156 were sorted and analyzed by FISH for the presence of trisomy 8 using a specific probe for chromosome 8. Leukemic status of MDCs and PDCs isolated from the blood of AML patients.MDCs from patient UPN156 were sorted and analyzed by FISH for the presence of trisomy 8 using a specific probe for chromosome 8. This result is representative of 4 experiments performed with MDCs and PDCs isolated from 4 different patients. Mohamad Mohty et al. Blood 2001;98: ©2001 by American Society of Hematology

6 Maturation capacities of MDCs and PDCs isolated from patients after in vitro culture.(A,C) The 2 DC subsets CD11c+ILT3+ (MDCs) and CD11c−ILT3+ (PDCs) were sorted from the blood of healthy individuals or (B,D) from leukemic patients and analyzed by flow cyto... Maturation capacities of MDCs and PDCs isolated from patients after in vitro culture.(A,C) The 2 DC subsets CD11c+ILT3+ (MDCs) and CD11c−ILT3+ (PDCs) were sorted from the blood of healthy individuals or (B,D) from leukemic patients and analyzed by flow cytometry after 72 hours of culture with either GM-CSF, IL-4, and CD40L (MDCs) or IL-3 and CD40L (PDCs) for their expression of DC marker CD83 and costimulatory molecules. (A) MDCs and (C) PDCs isolated from healthy volunteers acquired the expression of CD83 and expressed the costimulatory molecules (CD80 and CD86). (B) In leukemic patients, the MDC subset could acquire CD83 and the costimulatory molecules CD80 and CD86. (D) PDCs from leukemic patients never acquired CD83 or costimulatory molecules. Open histograms represent cells stained with isotype-matched control mAbs. Results indicated are representative of those obtained from 4 healthy donors and 5 patients. Mohamad Mohty et al. Blood 2001;98: ©2001 by American Society of Hematology

7 T-cell stimulatory capacity of MDCs and PDCs isolated from AML patients.MDCs and PDCs were sorted from the blood of healthy individuals or leukemic patients and cultured for 3 days in the presence of GM-CSF, IL-4, and CD40L (MDCs) or IL-3 and CD40L (PDCs). T-cell stimulatory capacity of MDCs and PDCs isolated from AML patients.MDCs and PDCs were sorted from the blood of healthy individuals or leukemic patients and cultured for 3 days in the presence of GM-CSF, IL-4, and CD40L (MDCs) or IL-3 and CD40L (PDCs). (A) Allostimulatory activity of 3-day–cultured MDCs and PDCs was monitored by their ability to induce the proliferation of CD4+ naive T cells. The ratio between the proliferative response measured by [3H]thymidine incorporation of 105 T cells induced by 3 × 103 leukemic MDCs and PDCs and normal MDCs and PDCs is represented. Results are represented as the mean of the ratio obtained from 4 experiments performed with DCs isolated from patients UPN109, UPN156, UPN223, and UPN90. (B) Allostimulatory activity of freshly isolated and 3-day–cultured PDCs. Graded numbers of freshly isolated PDCs from healthy donor (▵) or patient UPN109 (▴), or 3-day–cultured PDCs from healthy donor (○) or patient UPN109 (●), were cocultured with 105 CD4+naive T cells for 6 days. CD14+ monocytes (purified by magnetic separation using CD14 microbeads; Myltenyi Biotec GmbH, Bergisch Gladbach, Germany) from healthy donor (▪) were prepared and used as control stimulators. Representative data of 3 experiments are shown. Mohamad Mohty et al. Blood 2001;98: ©2001 by American Society of Hematology

8 IFN-α production by PDCs isolated from healthy donors and AML patients after in vitro stimulation with HSV.PDCs were sorted by FACS and stimulated with HSV without additional cytokine. IFN-α production by PDCs isolated from healthy donors and AML patients after in vitro stimulation with HSV.PDCs were sorted by FACS and stimulated with HSV without additional cytokine. Results represent the mean of cytokine concentrations contained in the culture supernatant obtained from 2 different donors and patients UPN90, UPN109, UPN156, UPN223, and UPN243. Mohamad Mohty et al. Blood 2001;98: ©2001 by American Society of Hematology


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