A Rapid Culture Technique Produces Functional Dendritic‐Like Cells from Human Acute Myeloid Leukemia Cell Lines

Abstract

Most anti-cancer immunotherapeutic strategies involving dendritic cells (DC) as vaccines rely upon the adoptive transfer of DC loaded with exogenous tumour-peptides. This study utilized human acute myeloid leukemia (AML) cells as progenitors from which functional dendritic-like antigen presenting cells (DLC) were generated, that constitutively express tumour antigens for recognition by CD8+ T cells. DLC were generated from AML cell lines KG-1 and MUTZ-3 using rapid culture techniques and appropriate cytokines. DLC were evaluated for their cell-surface phenotype, antigen uptake and ability to stimulate allogeneic responder cell proliferation, and production of IFN-γ; compared with DC derived from normal human PBMC donors. KG-1 and MUTZ-3 DLC increased expression of CD80, CD83, CD86, and HLA-DR, and MUTZ-3 DLC downregulated CD14 and expressed CD1a. Importantly, both KG-1 and MUTZ-3-derived DLC promoted proliferation of allogeneic responder cells more efficiently than unmodified cells; neither cells incorporated FITC-labeled dextran, but both stimulated IFN-γ production from responding allogeneic CD8+ T cells. Control DC produced from PBMC using the FastDC culture also expressed high levels of critical cell surface ligands and demonstrated good APC function. This paper indicates that functional DLC can be cultured from the AML cell lines KG-1 and MUTZ-3, and FastDC culture generates functional KG-1 DLC.

Human leukocyte antigen class II quantification by targeted mass spectrometry in dendritic-like cell lines and monocyte-derived dendritic cells

“…Consistent with a previous published results [34][35][36][37][38][39][40][41] , we confirmed an increase of csHLADR following TNF and LPS treatment for the monocytic-like cell line MUTZ-3, where both stimulants elicited a response of similar magnitude. This is also consistent with the increase in allogenic T cell-activation potential by induced-MUTZ-3 found by T cell proliferation assays 38 . However, our assessment of total HLA-II abundance by mass spectrometry showed a larger effect in stimulated MUTZ-3 cells, suggesting that an accumulation of HLA-α chain may occur at the intracellular compartments.…”

“…Furthermore, signal transduction upon different maturation signals may be also affected by cell cycle phase and receptor-mediated responses at the single-cell level. Overall, these findings are consistent with the proinflammatory response by MUTZ-3 differentiated into DCs in cytokine production and chemokines as well as the immunophenotypic profile reported, which maturation more closely recapitulates that of DCs [34][35][36]38,41 .We have shown that HLA-DRA1 protein is the most abundant α-chain variant (> 90%), followed by -DPA1 (3-7%) and -DQA1 (< 1%) when detected, irrespective of the HLA-II haplotype in dendritic-cell lines KG-1 and MUTZ-3. This was also observed in monocyte-derived DCs, where HLA-DRA1 levels were consistently higher than -DPA1, and -DPA1 was consistently higher than DQA1.…”

“…Briefly, KG-1 cells were cultured in RPMI 1640 Medium (RPMI; Gibco, Thermo Fisher Scientific, MA, USA) supplemented with 20% (v/v) Fetal Bovine Serum (FBS; Hyclone, Logan, USA) and penicillin/streptomycin (100 U/mL); MUTZ-3 cells were cultured with α-Minimum Essential Medium (α-MEM; Gibco, Thermo Fisher Scientific) supplemented with 20% (v/v) FBS, 100 U/mL penicillin/streptomycin and 25 IU/mL GM-CSF (R&D systems, Minneapolis, USA). MUTZ-3 maturation was stimulated as previously described 34 , 38 , 41 . Briefly, 2–4 × 10 5 cells/mL were seeded in complete media with 50 ng/mL GM-CSF and 20 ng/mL Interleukin-4 (IL-4, R&D systems) for 7 days.…”

Quality control of HLA-DR molecules by the lysosomal aspartyl protease, cathepsin D

“…Priess is a DRB1*04:01-homozygous lymphoblastoid cell line (84). KG-1 is an acute myleloid leukemia cell line (85) with features resembling immature DCs (86,87), expressing DRB1*11 and *14 (88). Cells were grown at 37 ˚C in a humidified incubator, in RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and penicillin/streptomycin, except for KG-1 cells, which were grown in IMDM with 20% Monocyte-derived dendritic cell culture Monocyte-derived dendritic cells were derived from healthy donor blood as described (45) with modifications as follows.…”

Can Dendritic Cell Vaccination Prevent Leukemia Relapse?

“…In AML, this approach is complicated by the difficulty to generate DCs from every patient [42]. To circumvent this, AML cell lines such as MUTZ-3 [92] and DCOne [93] have been used to generate allogeneic leukemic-DCs. Notably, DCP-001, a novel allogeneic DC vaccine generated from the DCOne AML cell line has shown remarkable results in a cohort of elderly advanced-stage AML patients, with 6/12 patients unexpectedly surviving over 6 months [93].…”