A Unique Immunotherapeutic Atrategy by Using a Leukemic Plasmacytoid Dendritic Cell Line (PMDC05) as Potent Antigen Presenting Cells.
Authors: Nakamura Takeshi , Narita Miwako , Yamahira Akie , Watanabe Norihiro , Tochiki Nozomi , Saitoh Anri , Kaji Masami , Taniguchi Tomoyo , Hashimoto Shigeo , Furukawa Tatsuo , Toba Ken , Fuse Ichiro , Aizawa Yoshifusa , Takahashi Masuhiro 
Published: October 2019

Abstract
Abstract 5117
Although cellular immunotherapy based on antigen-specific cytotoxic T lymphocytes (CTLs) against tumors including leukemia and severe infections is a promising strategy, one of the pivotal issues is a hardship in constant supply of high quality antigen presenting cells for generating autologous CTLs against antigens associated with tumor cells or pathogens. We established a leukemic plasmacytoid dendritic cell (pDC) line (PMDC05) with potent antigen presenting capacity from leukemia cells of a HLA-A*0201/*2402 patient with pDC acute leukemia. We investigated whether PMDC05 could be efficiently used for generating CTLs specific for antigens of leukemia cells or pathogens and whether PMDC05 could be applicable for cellular immunotherapy for tumors and infections. PMDC05, which grew in the absence of feeder cells, was positive for CD4, CD56, CD33, HLA-DR, CD123 (IL-3Rα) and CD86 in the absence of lineage markers. mRNA of TLR1, TLR2, TLR4, TLR7 and TLR9 were clearly expressed and among these TLRs, TLR7 was prominent. Transcripts of preTα, SpiB, MX1, IRF1, IRF7 and IFN-α14 were markedly expressed in PMDC05 and those of λ-like 14.1, IL-12p35 or IL-12p40 were also expressed. By culturing PMDC05 with IL-3, CpG-B or LPS, the expression of CD1a, CD80, CD83, CD86 or HLA-DR was remarkably enhanced. Transcripts of IRF1, IRF7, IFN-α14, IL-12p35 and IL-12p40 were increased by culturing with CpG-A, CpG-B, influenza virus or LPS. Production of IFN-α and IL-12p75 was enhanced by the stimulation with CpG-A and LPS, respectively. PMDC05 possessed a considerable antigen presenting ability to naïve T cells, which was enhanced by culturing with IL-3 or influenza virus and especially by LPS, and the ability to uptake lucifer yellow. These findings revealed that PMDC05 is a unique leukemic pDC cell line with potent antigen presenting ability. Therefore, we tried to generate WT1 or CMV-specific CTLs by using PMDC05 pulsed with relevant antigen as antigen presenting cells. PMDC05, which was stimulated with 0.1 μg/ml LPS and loaded with 10 μg/mL WT1 peptides (HLA-A*2402-restricted, modified-type 9-mer peptide; CYTWNQMNL) or CMV pp65 peptides (HLA-A*2402-restricted, 9-mer peptide; QYDPVAALF) for 24 hours, was irradiated (60 Gy) and co-cultured with allogeneic CD8+ T cells purified from peripheral blood mononuclear cells (PB-MNCs) of HLA-A*2402+ healthy donor (PMDC05:CD8+ Tcells = 1:10). 50 U/ml IL-2 was added to the co-culture at day 2, and IL-2 as well as 10 ng/ml IL-7 were added every 3 days thereafter. Burkitt lymphoma cell line, Daudi, was used as control of PMDC05 in the same manner. PB-MNCs, which were presumed to contain regulatory T (Treg) cells, were also co-cultured with PMDC05 or Daudi. Induction of WT1 or CMV-specific CTLs was evaluated by flow cytometry analysis using HLA-A*2402 WT1 tetramer or CMV tetramer every week. Although Daudi pulsed with WT1 peptides could not induce WT1 tetramer+CD8+ T cells during the co-culture up to 7 weeks, PMDC05 began to induce WT1 tetramer+CD8+ T cells at week 4 and the percentage of WT1 tetramer+ T cells in CD8+ T cells rose to more than 75% at week 7. Likewise, CMV tetramer+CD8+ T cells were amplified in CD8+ T cells co-cultured with CMV peptide-pulsed PMDC05 but not in those with Daudi. Although CMV tetramer+CD8+ T cells were generated in MNCs co-cultured with PMDC05 pused with CMV peptides, WT1 tetramer+CD8+ T cells were not observed in in MNCs co-cultured with PMDC05. These data suggested that PMDC05 could be efficiently used for generating CTLs specific for antigens of tumors or pathogens and could be applicable for cellular immunotherapy for tumors and infections. In addition, it was suggested that generation of antigen-speific CTLs might be affected by co-existing Treg cells in some tumor specific antigens.

Disclosures
No relevant conflicts of interest to declare.