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. 2005 Oct;54(10):953-70.
doi: 10.1007/s00262-004-0657-y. Epub 2005 Mar 24.

Serum-free generation and quantification of functionally active Leukemia-derived DC is possible from malignant blasts in acute myeloid leukemia and myelodysplastic syndromes

S Kufner  1 , R Pelka Fleischer, T Kroell, C Schmid, H Zitzelsberger, H Salih, F de Valle, W Treder, H M Schmetzer
Affiliations

Serum-free generation and quantification of functionally active Leukemia-derived DC is possible from malignant blasts in acute myeloid leukemia and myelodysplastic syndromes

S Kufner et al. Cancer Immunol Immunother. 2005 Oct.

Abstract

Functional dendritic cells (DC) are professional antigen presenting cells (APC) and can be generated in vitro from leukemic cells from acute myeloid leukemia AML patients, giving rise to APC of leukemic origin presenting leukemic antigens (DC(leu)). We have already shown that DC can be successfully generated from AML and myeloplastic syndromes (MDS) cells in serum-free 'standard' medium (X-vivo + GM-CSF + IL-4 +TNFalpha + FL) in 10-14 days. In this study, we present that DC counts generated from mononuclear cells (MNC) varied between 20% (from 55 MDS samples), 34% (from 100 AML samples) and 25% (from 38 healthy MNC samples) medium. Between 53% and 58% of DC are mature CD83+ DC. DC harvests were highest in monocytoid FAB types (AML-M4/M5, MDS-CMML) and independent from cytogenetic risk groups, demonstrating that DC-based strategies can be applied for patients with all cytogenetic risk groups. Proof of the clonal derivation of DC generated was obtained in five AML and four MDS cases with a combined FISH/immunophenotype analysis (FISH-IPA): The clonal numerical chromosome aberrations of the diseases were regularly codetectable with DC markers; however, not with all clonal cells being convertible to leukemia-derived DC(leu) (on average, 53% of blasts in AML or MDS). To the contrary, not all DC generated carried the clonal aberration (on average, 51% of DC). In 41 AML and 13 MDS cases with a suitable antigen expression, we could confirm FISH-IPA data by Flow cytometry: although DC(leu) are regularly detectable, on average only 57% of blasts in AML and 64% of blasts in MDS were converted to DC(leu). After coculture with DC in mixed lymphocyte reactions (MLR), autologous T cells from AML and MDS patients proliferate and upregulate costimulatory receptors. The specific lysis of leukemic cells by autologous T cells could be demonstrated in three cases with AML in a Fluorolysis assay. In six cases with only few DC(leu) or few vital T cells available after the DC/MLR procedure, no lysis of allogeneic or autologous leukemic cells was seen, pointing to the crucial role of both partners in the lysis process. We conclude: (1) the generation of DC is regularly possible in AML and also in MDS under serum-free conditions. (2) Clonal/leukemia-derived DC(leu) can be regularly generated from MDS and AML-MNC; however, not with all blasts being converted to DC(leu) and not all DC generated carrying leukemic markers. We recommend to select DC(leu) for vaccinations or ex vivo T-cell activations to avoid contaminations with non-converted blasts and non-leukemia-derived DC and to improve the harvest of specific, anti-leukemic T cells. DC and DC-primed T cells could provide a practical strategy for the immunotherapy of AML and MDS.

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Figures

Fig. 1
Fig. 1
DC from AML- and MDS-MNC were grown in GM-CSF, IL-4, TNFα and FL containing Xvivo media for 10–14 days and characterized (a, b) or used to stimulate autologous T cells in MLR in RPMI 1640 media and 10% FCS for 5 days (c). a Morphology of cytospinned DC in a case of AML-M4 after Giesma stain and photograph (x800 fold magnification) shows characteristics of DC: irregular shape and cytoplasmic projections. b left side: Flow-cytometric analyses of AML-M5-MNC before and after the culture procedure demonstrate high blast (7.1+) counts and low CD83 counts in the MNC fraction and high proportions of double stained cells in the DC fraction, thereby proving the leukemic origin of these DC. right side: FISH-IPA analysis, a combined FISH and surface marker assay, enables a distinction of cells coexpressing DC-marker CD1a and the clonal trisomy 8 from non-clonal DC and clonal CD1a-negative cells in the case of MDS-RAEB, thereby proving the leukemic origin of these DC. c Flow cytometric analysis of T cells after (left side) and before (right side) coculture with autologous DC in a case of AML-M5 demonstrate a T-cell activation with respect to proliferation (coexpression of CD71 on CD3+ T-cells) and expression of costimulary ligands (e.g. CD28): After 5 days, an increased Forward Scatter of T cells as well as an expansion of proliferating T cells coexpressing CD28 can be seen
Fig. 2
Fig. 2
Viability and degree of maturation of DC from AML and MDS-MNC or leukemic cell lines grown in GM-CSF, IL-4, TNFα and FL containing Xvivo media for 10–14 days (AML/MDS/cell lines) or 7 days (healthy donors). Proportions of immunocytologically detectable blasts in AML or MDS (CD34+ or CD117+ cells in MDS)—MNC and proportions of DC generated from the total MNC-fraction as well as proportions of mature (CD83+ or CD14− DC) or viable DC in DC fractions from healthy, AML or MDS samples (a) or from cell lines (b) are given
Fig. 3
Fig. 3
DC from AML- and MDS-MNC at first diagnosis were grown in GM-CSF, IL-4, TNFα and FL containing Xvivo media for 10–14 days and separated according to FAB types (a) or cytogenetic risk groups (b). Proportions of DC generated are given: DC harvests were highest in monocytoid FAB types, but independent of the cytogenetic risk group
Fig. 4
Fig. 4
DC from AML- and MDS-MNC or cell lines were grown in GM-CSF, IL-4, TNFα and FL containing Xvivo media for 10–14 days (healthy donors for 7 days), characterized (a) and used to stimulate autologous T cells in an MLR in RPMI 1640 media and 10% FCS for 5 days (b). a Proportions of DC coexpressing costimulatory ligands are given b Percentual changes of autologous T cells coexpressing costimulatory receptors after the 5-day coculture MLR procedure (‘[Tauto] d5’) compared to non-DC-stimulated controls (‘[Tauto +DC] d5’) are given. Moreover, a comparison of the T-cell stimulatory effect of MNC (‘[Tauto + MNC] d5’) compared to DC after a 5-day coculture with autologous T cells (‘[Tauto +DC] d5’) is given
Fig. 5
Fig. 5
DC from AML- and MDS-MNC were grown in GM-CSF, IL-4, TNFα and FL containing Xvivo media for 10–14 days and the leukemic/clonal derivation proven by FISH-IPA or Flow cytometry. a Clonal derivation of DC generated from five cases with AML (left side) and four cases with MDS (right side) was proven by codetection of the clonal numerical chromosome aberration with DC (FISH-IPA-analysis). b Quantitative analyses by FISH-IPA or Flow cytometry show that 18–80% of clonal cells in the five cases given are converted to DC carrying the clonal marker (left side); that on average, 46% of leukemic cells in AML (n=32) and 60% of CD34 or CD117+ cells in MDS (n=12) are converted to DCleu (middle). In contrast, 31–61% of DC created from AML and MDS are of clonal origin in the five cases given (right side). c Dot-plot analyses generated in five cases with AML or MDS show that distinct blast populations can be detected in MNC in the ‘SSC/blast-marker’ plot (first row), and that those blast populations do not express DC-markers (second row). After 10–14 days of culture in GM-CSF, IL-4, TNFα and FL containing Xvivo medium, the blast populations gain an increased SSC and an expression of DC markers (third and fourth row). Statistical evaluations allow the exact quantification of blasts which were converted to DCleu (third and fourth row).
Fig. 5
Fig. 5
DC from AML- and MDS-MNC were grown in GM-CSF, IL-4, TNFα and FL containing Xvivo media for 10–14 days and the leukemic/clonal derivation proven by FISH-IPA or Flow cytometry. a Clonal derivation of DC generated from five cases with AML (left side) and four cases with MDS (right side) was proven by codetection of the clonal numerical chromosome aberration with DC (FISH-IPA-analysis). b Quantitative analyses by FISH-IPA or Flow cytometry show that 18–80% of clonal cells in the five cases given are converted to DC carrying the clonal marker (left side); that on average, 46% of leukemic cells in AML (n=32) and 60% of CD34 or CD117+ cells in MDS (n=12) are converted to DCleu (middle). In contrast, 31–61% of DC created from AML and MDS are of clonal origin in the five cases given (right side). c Dot-plot analyses generated in five cases with AML or MDS show that distinct blast populations can be detected in MNC in the ‘SSC/blast-marker’ plot (first row), and that those blast populations do not express DC-markers (second row). After 10–14 days of culture in GM-CSF, IL-4, TNFα and FL containing Xvivo medium, the blast populations gain an increased SSC and an expression of DC markers (third and fourth row). Statistical evaluations allow the exact quantification of blasts which were converted to DCleu (third and fourth row).
Fig. 6
Fig. 6
DC from AML- and MDS-MNC or healthy donors were grown in GM-CSF, IL-4, TNFα and FL containing Xvivo media for 10–14 days (healthy donors for 7 days), characterized (a) and used to stimulate autologous or allogeneic T cells in an MLR in RPMI 1640 media and 10% FCS for 5 days. a Percentual changes of autologous T cells coexpressing proliferation markers after a 5-day coculture in MLR cultures (‘[Tauto] d5’) compared to non-DC-stimulated controls (‘[Tauto +DC] d5’) are given. Moreover, a comparison of the T-cell stimulatory effect of MNC (‘[Tauto +MNC] d5’) compared to DC after 5-day coculture with autologous T cells (‘[Tauto +DC] d5’) is given. b DC-stimulated autologous T cells from three AML-patients grown in an MLR (RPMI medium, 10% FCS) were cocultured with naïve autologous blasts for 3 h. Specific lyses of blasts after coculture with T cells compared to controls, evaluated by a ‘Fluorolysis assay’, are given (upper part). E: Effector cells (activated autologous (auto) or allogeneic (allo) T-cells); T: Target cells (naïve blasts). In parallel, the specific lysis of other autologous target cells from AML patients (open square, open diamond, –) or healthy donors (x, open triangle) by autologous T cells from AML patients (open square, open diamond, –) or healthy donors (x, open triangle) evaluated by a ‘Fluorolysis assay’ are given (lower part)
Fig. 7
Fig. 7
DC from AML- and MDS-MNC or healthy donors were grown in GM-CSF, IL-4, TNFα and FL containing Xvivo media or in ‘MCM-Mimic’ (Xvivo+GM-CSF, IL-4, TNFα, FL, IL-6, IL-1β, PGE2) for 10–14 days (healthy donors for 7 days). Percent of mature, CD83+ DC as well as CCR7+ DC are given
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