Effective and Successful Quantification of Leukemia-Specific Immune Cells in AML Patients’ Blood or Culture, Focusing on Intracellular Cytokine and Degranulation Assays

Abstract

Novel (immune) therapies are needed to stabilize remissions or the disease in AML. Leukemia derived dendritic cells (DCleu) can be generated ex vivo from AML patients’ blasts in whole blood using approved drugs (GM-CSF and PGE-1 (Kit M)). After T cell enriched, mixed lymphocyte culture (MLC) with Kit M pretreated (vs. untreated WB), anti-leukemically directed immune cells of the adaptive and innate immune systems were already shown to be significantly increased. We evaluated (1) the use of leukemia-specific assays [intracellular cytokine production of INFy, TNFa (INCYT), and degranulation detected by CD107a (DEG)] for a detailed quantification of leukemia-specific cells and (2), in addition, the correlation with functional cytotoxicity and patients’ clinical data in Kit M-treated vs. not pretreated settings. We collected whole blood (WB) samples from 26 AML patients at first diagnosis, during persisting disease, or at relapse after allogeneic stem cell transplantation (SCT), and from 18 healthy volunteers. WB samples were treated with or without Kit M to generate DC/DCleu. After MLC with Kit M-treated vs. untreated WB antigen-specific/anti-leukemic effects were assessed through INCYT, DEG, and a cytotoxicity fluorolysis assay. The quantification of cell subtypes was performed via flow cytometry. Our study showed: (1) low frequencies of leukemia-specific cells (subtypes) detectable in AML patients’ blood. (2) Significantly higher frequencies of (mature) DCleu generable without induction of blast proliferation in Kit M-treated vs. untreated samples. (3) Significant increase in frequencies of immunoreactive cells (e.g., non-naive T cells, Tprol) as well as in INCYT/DEG ASSAYS leukemia-specific adaptive—(e.g., B, T(memory)) or innate immune cells (e.g., NK, CIK) after MLC with Kit M-treated vs. untreated WB. The results of the intracellular production of INFy and TNFa were comparable. The cytotoxicity fluorolysis assay revealed significantly enhanced blast lysis in Kit M-treated vs. untreated WB. Significant correlations could be shown between induced leukemia-specific cells from several lines and improved blast lysis. We successfully detected and quantified immunoreactive cells at a single-cell level using the functional assays (DEG, INCYT, and CTX). We could quantify leukemia-specific subtypes in uncultured WB as well as after MLC and evaluate the impact of Kit M pretreated (DC/DCleu-containing) WB on the provision of leukemia-specific immune cells. Kit M pretreatment (vs. no pretreatment) was shown to significantly increase leukemia-specific IFNy and TNFa producing, degranulating cells and to improve blast-cytotoxicity after MLC. In vivo treatment of AML patients with Kit M may lead to anti-leukemic effects and contribute to stabilizing the disease or remissions. INCYT and DEG assays qualify to quantify potentially leukemia-specific cells on a single cell level and to predict the clinical course of patients under treatment.