Identification of Dendritic Cell Precursors in the Culture of Bone Marrow with Hematopoietic Cytokines

Abstract

Dendritic cells (DCs), key sentinels, play crucial parts in a variety of immune responses by capturing and presenting antigens to naïve T cells and by producing the subsequent T cell activation or tolerance. In vitro culture systems of DCs have been widely used to study DC biology and immunology. The culture of bone marrow (BM) in the presence of a hematopoietic cytokine, Fms-like tyrosine kinase 3 ligand (FLT3L), produces plasmacytoid DCs (pDCs) and classical DCs (cDCs) showed phenotype similar to steady state DCs, whereas the culture of BM with granulocyte-macrophage colony-stimulating factor (GM-CSF) generates DCs and macrophages considered to possess inflammatory phenotypes. In the present study, the culture of BM was examined with both cytokines, i.e., FLT3L and GM-CSF in combination and 4 different subsets were identified in CD11c⁺MHC II+ cells. Meanwhile, in the culture of BM with FLT3L, MHC IIhi classical DC2s (cDC2s) were identified as a heterogeneous subset with the additional use of hematopoietic markers. CD11c⁺MHC IIhi cells in FLT3L conditioned BM culture were identified as at least three different subsets and a novel population of CD11c⁺MHC IIhi cells were discovered which are possess a limited capacity of antigen-presenting activity and superior capacity of taking up antigens, suggesting functionally immature DCs. This novel population from the BM culture with FLT3L was isolated and evaluated for its potential to differentiate to DCs in the BM culture containing both FLT3L and GM-CSF. As a result, the novel immature DC-like cells in CD11c⁺MHC IIhi cells from BM culture with FLT3L can be activated and differentiate into mature or activated DCs, which means that the novel population is an immediate precursor cell for DCs. Furthermore, the morphology of each subset in CD11c⁺MHC IIhi cells was illustrated and showed distinct characteristics. Consequently, the presence of the novel population of DC precursors in the in vitro culture system of DCs was demonstrated. At the transcriptional level, the newly discovered subsets in CD11c⁺MHC IIhi cells were analyzed by RNA sequencing. In addition, to figure out the ontogeny of each subset, hematopoietic progenitors were cultured in the same condition that yield the MHC IIhi cell subsets. Collectively, in the current study, the novel populations and the novel DC precursors generated from BM culture in presence of FLT3L were identified for the first time. In summary, heterogeneity in CD11c+MHC II+ FLT3L-derived cDCs were unveiled. 수지상세포는 다양한 면역 작용에 있어 중요한 역할을 하는 면역세포로서, 체내에서 항원을 수집하고 처리하여 T 세포에 전달하는 전문 항원제시세포이다. 수지상세포의 체외 배양 시스템은 수지상세포 연구에 널리 쓰이는 바 있으며 수지상세포의 분화에 중요한 조혈 사이토카인인 과립구 대식세포 콜로니 자극인자 (GM-CSF)와 FMS-유사 티로신 키나제 3 리간드 (FLT3L)가 이에 사용된다. GM-CSF 조건 하에 골수세포를 배양할 경우 염증환경에서 생성되는 수지상세포와 대식세포가 생성된다. 반면, FLT3L의 존재 하에 배양된 골수세포로부터는 형질세포양 수지상세포 (plasmacytoid DCs, pDCs)와 정통 수지상세포 (classical DCs, cDCs)가 생성된다. 본 연구에서는 두 사이토카인을 사용하여 배양한 골수세포 배양에서 CD11c와 MHC II 표지분자를 발현하는 새로운 아형세포를 발굴하였다. 특히 FLT3L 골수 배양에서는 기존에 정통 수지상세포 2 아형으로 알려졌던 세포집단에서 CD11c와 MHC II 표지분자를 높게 발현하면서 항원제시능력이 낮은 수지상세포의 전구세포와, 항원제시능력이 높으면서 T 세포의 분화를 잘 유도할 수 있는 활성 수지상세포2 아형을 발굴하였다. 이 새로운 세포집단을 각각 분리하여 세포의 형태를 관찰하고 수지상세포로서의 기능을 평가하였으며, 수지상세포의 전구세포의 경우 여러 자극인자를 사용하여 수지상세포로의 분화능을 평가하였다. 또한 새로 발굴한 수지상세포 아형세포들의 유래와 개체발생을 확인하기 위해, 수지상세포의 전구세포로 알려진 조혈 전구세포들을 분리 배양하여 이를 확인하였다. 결과적으로, 수지상세포의 체외 배양을 통해 수지상세포의 전구세포를 생성할 수 있음을 증명하였다.