論文の公開元へA Novel CD135⁺ Subset of Mouse Monocytes with a Distinct Differentiation Pathway and Antigen-Presenting Properties
概要
The Journal of Immunology
A Novel CD135+ Subset of Mouse Monocytes with a Distinct
Differentiation Pathway and Antigen-Presenting Properties
Naoka Kamio,*,†,‡,1 Asumi Yokota,‡,§,1 Yuichi Tokuda,{ Chie Ogasawara,‖ Masakazu Nakano,{
Miki Nagao,* Kei Tashiro,{ Taira Maekawa,†,# Nobuyuki Onai,‖ and Hideyo Hirai*,†,‡
endritic cells (DCs) play a central role in the immune system by connecting innate and adaptive immunity. Conventional DCs (cDCs) sense and ingest external Ags and
present them on MHC class II, which activates naive T lymphocytes
(14). In addition to cDCs, monocyte-derived DCs (moDCs) and
plasmacytoid DCs (pDCs) are present in both mice and humans
(57). All of these DC types are crucial for immune responses and maintenance of homeostasis. moDCs are induced in response to microbial
infections and regulate the function of CD41 or CD81 T lymphocytes by
presenting ingested Ags (79). pDCs, originating from both myeloid and
lymphoid progenitors (10), secrete type I IFN upon their activation by signals mediated through TLR7 and TLR9 during viral infections (11, 12).
DCs and monocytes/macrophages compose the mononuclear phagocyte system (MPS), and they are generally considered to originate from
common progenitor cells in the bone marrow (BM), although arguments
still remain (1, 1315). Macrophage DC progenitors (MDPs) are considered to reside at the top of the hierarchy within the MPS, giving rise
to both monocyte and DC lineages (16). Common DC progenitors
(CDPs) (17) and common monocyte progenitors (cMoPs) (18) have
been identified as exclusive progenitors for DCs and monocytes, respectively. MDPs and CDPs, both of which are progenitors of cDCs and
pDCs, express Flt3 (also known as CD135) on their surface. Flt3
D
mRNA was detected in short-term hematopoietic stem cells, and the
expression peaked in multipotent progenitors (19). Progenitor cells
retaining surface CD135 expression have the potential to give rise to
DCs, whereas progenitors, which no longer express CD135, lose their
ability to differentiate into DCs. Also, the number of DCs is significantly
reduced in the absence of Flt3 signaling, suggesting a critical role for the
Flt3 ligand (Flt3L)Flt3 interaction in the development of DC lineages
(2, 20, 21).
moDCs are distinct from other DCs as they are generally considered to originate from monocytes rather than from undifferentiated
progenitors (6, 22). Under inflammatory conditions, monocytes
acquire CD11c and MHC class II expression, which indicates their
differentiation into moDCs (2326). In addition, large numbers of
moDCs can be obtained by in vitro culture of monocytes (7, 27,
28). Therefore, moDCs have been widely used for intensive characterization of DCs and have been used in immunotherapies (29, 30).
Accumulating evidence has shown that monocytes are a heterogeneous population in mice and humans (14, 31, 32). In mice, monocytes
are classified based on the expression level of Ly6C. Ly6Chigh and
Ly6Clow monocytes represent “classical” and “nonclassical” monocytes, respectively (33, 34). In addition, recent technical advances,
including single-cell analysis, have further extended our understanding
*Department of Clinical Laboratory Medicine, Kyoto University Hospital, Kyoto, Japan;
†
Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto,
Japan; ‡Laboratory of Stem Cell Regulation, School of Life Sciences, Tokyo University
of Pharmacy and Life Sciences, Tokyo, Japan; xDivisions of Pathology and Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center,
OH; {Department of Genomic Medical Sciences, Kyoto Prefectural University of Medicine, Kyoto, Japan; ‖Department of Immunology, Kanazawa Medical University, Japan;
and #Kyoto Prefectural Institute of Public Health and Environment, Kyoto, Japan
formal analysis, writing review and editing, project administration, and funding
acquisition.
The sequences presented in this article have been submitted to the DNA Data Bank of
Japan under accession numbers E-GEAD-338 and DRA009516.
1
Address correspondence and reprint requests to Dr. Hideyo Hirai, Laboratory of Stem
Cell Regulation, School of Life Sciences, Tokyo University of Pharmacy and Life
Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. E-mail address: hirai@
toyaku.ac.jp
ORCIDs: 0000-0001-6720-2158 (M. Nakano), 0000-0002-8886-6145 (M. Nagao).
The online version of this article contains supplemental material.
Received for publication January 12, 2021. Accepted for publication May 24, 2022.
Abbreviations used in this article: BM, bone marrow; cDC, conventional DC; cDC2,
cDC type 2; CDP, common DC progenitor; cMoP, common monocyte progenitor; DC,
dendritic cell; DC-SIGN, DC-specific ICAM-3grabbing nonintegrin; DTR, diphtheria
toxin receptor; Flt3L, Flt3 ligand; FPKM, fragments per kilobase of transcript per
million mapped reads; iNOS, inducible NO synthase; Lin, lineage; MDP, macrophage
DC progenitor; moDC, monocyte-derived DC; MPS, mononuclear phagocyte system;
NGS, next-generation sequencing; PB, peripheral blood; pDC, plasmacytoid DC; RNAseq, RNA sequencing.
N.K. and A.Y. contributed equally to this work.
This work was partly supported by KAKENHI Grants-in-Aid for Scientific Research
from the Japan Society for the Promotion of Science and University Grants 18K08354,
21K19386 and 21H02956 (to H.H.) and 21K08379 (to A.Y.).
N.K.: methodology, validation, formal analysis, investigation, writing original draft and
visualization; C.O.: investigation; Y.T., M. Nakano, and K.T.: validation, formal analysis, and
writing review and editing; A.Y.: validation and formal analysis, investigation, and writing
review and editing; M. Nagao and T.M.: supervision; N.O.: methodology, validation, formal
analysis, and writing review and editing; H.H.: conceptualization, methodology, validation,
www.jimmunol.org/cgi/doi/10.4049/jimmunol.2100024
Copyright © 2022 by The American Association of Immunologists, Inc. 0022-1767/22/$37.50
Downloaded from http://www.jimmunol.org/ at Kyoto Furitsu Ika Daigaku on September 20, 2022
The mononuclear phagocyte system (MPS), composed of monocytes/macrophages and dendritic cells (DCs), plays a critical role at
the interface of the innate and adaptive immune systems. However, the simplicity of MPS has been challenged recently by
discoveries of novel cellular components. In the current study, we identified the CD135+ subset of monocytes as a novel class of
APCs in mice. CD135+ monocytes were readily found in the bone marrow, spleen, and peripheral blood at steady state, and they expressed
markers specific to DCs, including MHC class II and CD209a, along with markers for monocytes/macrophages. In addition, this subset
phagocytosed bacteria and activated naive T lymphocytes, fulfilling the criteria for APCs. CD135+ monocytes were derived directly from
macrophage DC progenitors, not from common monocyte progenitors or other monocytes, suggesting that these are distinct from
conventional monocytes. These findings facilitate our understanding of the MPS network that regulates immune responses for host
defense. The Journal of Immunology, 2022, 209: 498509.
The Journal of Immunology
499
Finally, 4891 genes were selected for analyses based on the results from the
Cuffdiff option of Cufflinks under the following quality control conditions:
1) excluding genes where the “status” was not “OK,” 2) excluding genes
that showed an error value for “log2(fold_change),” 3) including genes that
showed “yes” for “significant,” 4) including genes that resulted in a |log2
(fold change)| $ 2.0, and 5) excluding genes when more than six samples
resulted in FPKM 5 0. For the heatmap analysis of myeloid transcription
factor genes, the heatmap images were drawn using the pheatmap package
after normalizing the FPKM values derived from 27 selected genes by using
the zFPKM R/Bioconductor package. The images of RNA-seq data were
drawn using the Integrative Genomics Viewer tool (43).
Materials and Methods
Giemsa staining
Mice
Cytospin specimens were stained using a Diff-Quik stain kit (Sysmex, Kobe,
Japan), a modified WrightGiemsa staining system (44). To analyze the dendrite formation by CD1351 monocytes, cells were incubated for 16 h in
RPMI 1640 cell culture medium supplemented with 10 ng/ml GM-CSF
(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 10% FCS,
50 mM 2-ME, and penicillin-streptomycin. Images were captured using an
Olympus BX43 microscope (Olympus, Tokyo, Japan) connected to a DP80
CCD camera, and processed using cellSens standard 1.12 software (Olympus).
C57BL/6 mice (CD45.21, 712 wk old) were purchased from CLEA Japan
(Tokyo, Japan) or Tokyo Laboratory Animals Science (Tokyo, Japan).
BALB/c mice (712 wk old) were purchased from Japan SLC (Shizuoka,
Japan). CD45.11 in C57BL/6 background mice were a gift from Shigekazu
Nagata (Osaka University, Osaka, Japan). All of the animal protocols were
approved by the Committee on Animal Research of the Kyoto University
Faculty of Medicine and by the Tokyo University of Pharmacy and Life Sciences Animal Use Committee.
Csf2−/− (38), Csf2rb−/− (39), Flt3l−/− (40), CD11cdiphtheria toxin
receptor (DTR) (41), and CX3CR1-GFP mice (42) were maintained in the
specific pathogen-free facility at Kanazawa Medical University, and all
experiments with these animals were also approved by the Institutional Animal Care Committees of Kanazawa Medical University.
Flow cytometric analysis and cell sorting
To obtain BM cells, tibia, femur, and humerus were flushed with PBS containing 2% FCS. Peripheral blood (PB) samples were collected from the
orbital venous plexus under anesthesia. To lyse RBCs, BM, spleen, or PB
cells were treated with Pharm Lyse reagent (BD Biosciences, San Jose, CA).
For surface marker staining, cells were stained with the fluorescent markerconjugated Abs listed in Supplemental Table I. Propidium iodide was used
to exclude dead cells. ...
