Dendritic Cell-Triggered Immune Activation Goes along with Provision of (Leukemia-Specific) Integrin Beta 7-Expressing Immune Cells and Improved Antileukemic Processes

Abstract

Integrin beta 7 (β7), a subunit of the integrin receptor, is expressed on the surface of immune cells and mediates cell-cell adhesions and interactions, e.g., antitumor or autoimmune reactions. Here, we analyzed, whether the stimulation of immune cells by dendritic cells (of leukemic derivation in AML patients or of monocyte derivation in healthy donors) leads to increased/leukemia-specific β7 expression in immune cells after T-cell-enriched mixed lymphocyte culture-finally leading to improved antileukemic cytotoxicity. Healthy, as well as AML and MDS patients' whole blood (WB) was treated with Kit-M (granulocyte-macrophage colony-stimulating factor (GM-CSF) + prostaglandin E1 (PGE1)) or Kit-I (GM-CSF + Picibanil) in order to generate DCs (DC_leu or monocyte-derived DC), which were then used as stimulator cells in MLC. To quantify antigen/leukemia-specific/antileukemic functionality, a degranulation assay (DEG), an intracellular cytokine assay (INTCYT) and a cytotoxicity fluorolysis assay (CTX) were used. (Leukemia-specific) cell subtypes were quantified via flow cytometry. The Kit treatment of WB (compared to the control) resulted in the generation of DC/DC_leu, which induced increased activation of innate and adaptive cells after MLC. Kit-pretreated WB (vs. the control) led to significantly increased frequencies of β7-expressing T-cells, degranulating and intracellular cytokine-producing β7-expressing immune cells and, in patients' samples, increased blast lysis. Positive correlations were found between the Kit-M-mediated improvement of blast lysis (vs. the control) and frequencies of β7-expressing T-cells. Our findings indicate that DC-based immune therapies might be able to specifically activate the immune system against blasts going along with increased frequencies of (leukemia-specific) β7-expressing immune cells. Furthermore, β7 might qualify as a predictor for the efficiency and the success of AML and/or MDS therapies.

Keywords: AML; MDS; immune therapy; integrin beta 7; leukemia-derived dendritic cells.

Figure 1

Generation of DC/DC_leu from (A1) leukemic and (B1) healthy WB with and without Kits. WB samples were cultured for 7 days with Kit-M or Kit-I or without added Kits as control. Results with Kit-M (DC(M)) or Kit-I (DC(I)) or without added Kits as control (DC(C)) are given. Mean frequencies ± standard deviation of DC subtypes in (A2) leukemic (AML/MDS) and (B2) healthy samples are given; n—number of cases. Differences were considered as significant (*) when p ≤ 0.05 and as highly significant (**) when p ≤ 0.005. Abbreviations of cell subpopulations are given in Table 1.

Figure 2

Composition of immune-reactive cells before and after T-cell-enriched MLC using (A) leukemic and (B) healthy WB with or without Kit pretreatment. Cells were analyzed via flow cytometry before and after 7 days of (T-cell-enriched) MLC with Kit-pretreated or untreated WB and IL-2. Cells before MLC from WB without added Kits as control (MLC(UC)), and cells after MLC, from WB pretreated with Kit-M (MLC(M)), Kit-I (MLC(I)) or without added Kits as control (MLC(CC)), are given. Mean frequencies ± standard deviation of immune-reactive cell subpopulations in (A) leukemic (AML/MDS) and (B) healthy samples are given; n—number of cases. Differences were considered as significant (*) when p ≤ 0.05 and as highly significant (**) when p ≤ 0.005. Double-sided arrows give (significant) differences between defined healthy and leukemic immune-reactive cell subtypes. Abbreviations of cell subpopulations are given in Table 1.

Figure 3

Composition of β7-expressing immune-reactive cells before and after T-cell-enriched MLC using (A) leukemic and (B) healthy WB with and without Kit pretreatment as stimulator cells. Cells were analyzed via flow cytometry before and after 7 days of (T-cell-enriched) MLC with Kit-pretreated or untreated WB and IL-2. Cells before MLC from WB without added Kits as control (MLC(UC)), and cells after MLC, from WB pretreated with Kit-M (MLC(M)), Kit-I (MLC(I)) or without added Kits as control (MLC(CC)), are given. Mean frequencies ± standard deviation of immune-reactive cell subpopulations in (A) leukemic (AML/MDS) and (B) healthy samples are given; n—number of cases. Differences were considered as significant (*) when p ≤ 0.05 and as highly significant (**) when p ≤ 0.005. Double-sided arrows give (significant) differences between defined healthy and leukemic immune-reactive cell subtypes. Abbreviations of cell subpopulations are given in Table 1.

Figure 4

Composition of β7-expressing degranulating or intracellular cytokine-producing immune-reactive cells in uncultivated WB and after T-cell-enriched MLC using (A) leukemic and (B) healthy WB with or without Kit pretreatment as stimulator cells. Degranulation and intracellular cytokine production were quantified via flow cytometry in untreated and uncultivated WB as well as after 7 days of (T-cell-enriched) MLC with Kit-pretreated or untreated WB and IL-2. Results without LAA or SEB stimulation are given. Uncultivated cells in WB and cultivated cells after MLC from WB pretreated with Kit-M (MLC(M)) or without added Kits as control (MLC(CC)) are given. Mean frequencies ± standard deviation of immune-reactive cell subpopulations in (A) leukemic (AML/MDS) and (B) healthy samples; n—number of cases. Differences were considered as significant (*) when p ≤ 0.05. Double-sided arrows give (significant) differences between defined healthy and leukemic immune-reactive cell subtypes. Abbreviations of cell subpopulations are given in Table 1.

Figure 5

Blastolytic potential of immune-reactive cells after T-cell-enriched MLC using leukemic WB with and without Kit pretreatment as stimulator cells. For the cytotoxicity assay, target and effector cells were coincubated for a total of 24 h. Results after 3 h and 24 h and the ‘best of’ values after coincubation are given. Results after MLC from WB pretreated with Kit-M (MLC(M)) or Kit-I (MLC(I)) or without added Kits as control (MLC(CC)) are given. Percentages of cases (A1) with achieved (vs. non-achieved) blast lysis and (B1) with improved (vs. non-improved) blast lysis are given. Mean frequencies ± standard deviation of (A2) lysed blasts (in cases with lysis) and (B2) lysis improvement (in cases with improved lysis) are given; n—number of cases. Differences were considered as significant (*) when p ≤ 0.05. Abbreviations of cell subpopulations are given in Table 1.

Figure 6

Composition of uncultivated β7-expressing immune-reactive cells in AML patients’ samples with patients subdivided into different groups at first diagnosis. Uncultured cells (MLC(UC)) were analyzed via flow cytometry. Mean frequencies ± standard deviation of β7-expressing immune-reactive cell subpopulations in patients with AML at first diagnosis with respect to patients’ (A) responses to chemotherapy and (B) allocation to cytogenetic ELN risk groups are given; n—number of cases. Abbreviations of cell subpopulations are given in Table 1.

Figure 7

Correlations of antileukemic functionality with frequencies of (leukemia-specific) β7-expressing immune-reactive cells before and after T-cell-enriched MLC using Kit-M-pretreated (vs. untreated as control) leukemic WB as stimulator cells. Cells were analyzed via flow cytometry before and after 7 days of (T-cell-enriched) MLC with Kit-pretreated or untreated WB and IL-2. Results of uncultured cells before MLC (MLC(UC)), and cells after MLC, from WB pretreated with Kit-M (MLC(M)) or without added Kits as control (MLC(CC)), are given. Lysis (improvement) is given as the best of value after MLC(M) (compared to MLC(CC)). (A) Clear positive correlation of frequencies of T_β7+/CD3⁺ with best blast lysis after MLC (with Kit-M-pretreated leukemic WB). (B) Significant positive correlation of frequencies of T_β7+/CD3⁺) with T_β7+IFNγ+/T_β7+ after MLC (with Kit-M-pretreated leukemic WB. (C) Significant positive correlation of frequencies of T_β7+107a+/T_β7 with improved blast lysis after MLC (with Kit-M-pretreated leukemic WB). r—correlation coefficient, p—significance, n—number of cases. Differences were considered as significant when p ≤ 0.05 and as highly significant when p ≤ 0.005. Abbreviations of cell subpopulations are given in Table 1.