Dendritic Cells Co-Developing from Human CD34+ Cells Following Incubation with Thrombopoietin and Tumor Necrosis Factor-α Phagocytose Self-Megakaryocytic Progenitors and Induce Autologous T-Cell Proliferation.
Authors: Saito Kunie , Hirokawa Makoto , Fukaya Hiroshi , Kawabata Yoshinari , Komatsuda AtAtsushi , Yamashita JuJunsuke , Sawada Ken-Ichi 
Published: April 2020

Abstract
Background. Thrombopoietin (TPO) and tumor necrosis factor-α (TNF-α) sustain differentiation and proliferation of CD34+ cells toward dendritic cells (DC) in the presence of multi-acting cytokines. Therefore, we hypothesized that stimulation of human CD34+ cells with TPO and TNF-α might co-develop megakaryocytic progenitors and DC, which may relate to the induction of immune tolerance and autoimmunity in megakaryopoiesis. Materials and Methods. Highly purified human CD34+ cells were cultured in liquid phase with TPO, with or without TNF-α, and induced to undergo megakaryocytic differentiation. We enumerated megakaryocytic progenitor cells using the specific markers CD41, CD42b and CD61, and DC using CD4, CD11c, CD80, CD83, CD86 and CD123. The character and roles of co-developing non-megakaryocytic cells in the presence of TNF-α were analyzed using fluorescent activating cell sorter, enzyme immunohistochemistry, confocal microscopy and autologous mixed lymphocyte reaction (AMLR). Results. When CD34+ cells were cultured for 7 days in the presence of TPO at 100 ng/ml, the generated cells predominantly expressed CD41 (95±2%), CD42b (54±12%) and CD61 (96±2%), while rarely expressed CD11c (1.6±1.3%), CD80 (0.1±0.1%), CD83 (0.8±0.6%) or CD86 (3.3±1.9%). In contrast, addition of TNF-α at 100 ng/ml significantly decreased cells expressing CD41 (3.0±0.6%), CD42b (3.3±1.0%) or CD61 (3.2±0.9%), but did not affect the number of total cells. In the presence of TNF-α, the generated cells expressed HLA class I (100%) and HLA class II (100%), and a substantial number of cells became positive for CD11c (37±1%), even costimulatory molecules, such as CD80 (2.4±1.9%), D83 (8±4%) and CD86 (18±7%). TNF-α induced apoptosis of megakaryocytic cells. Immature CD11c+ DC was physically associated with apoptotic and CD61+ cells and was capable of endocytosing CD61+ cells. All of CD11c+ cells co-expressed c-mpl, CD4 and CD123, and about a half of CD11c+ cells co-expressed CD86. Cells generated by TNF-α and TPO (DC: TPO+TNF-α) induced autologous T cell proliferation in AMLR assay, however, cells generated by TNF-α alone (DC: TNF-α) did not (Figure 1A). Immunophenotypic analysis of both populations showed the higher expression of co-stimulatory molecules such as CD80, CD83 and CD86 in cells generated by TNF-α and TPO (Figure 1B). Conclusions. Non-megakaryocytic cells co-generated from human CD34+ cells during megakaryocytic differentiation in the presence of TPO and TNF-α express DC phenotypes. The CD4+/CD11c+/CD123+ DC subset physically and selectively associates with developing immature megakaryocytic cells and then obtains and captures self-substances and are functional in AMLR. These findings suggest that DC generated from human CD34+ cells under megakaryocytic and inflammatory co-stimuli obtain a functional role and possibly leading to the antigen presentation to induce immunity or tolerance against megakaryocytic cells and/or platelets.
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