The developmental program of human dendritic cells is operated independently of conventional myeloid and lymphoid pathways

Abstract

Two distinct dendritic cell (DC) subsets, conventional DCs (cDCs) and plasmacytoid DCs (pDCs), have been shown to develop via either the myeloid or the lymphoid pathway in murine hematopoiesis. Lineage-specific phenotypes or functions of "myeloid" and "lymphoid" DCs, however, still remain elusive. Furthermore, such analysis has been particularly difficult in humans, due to lack of an assay system appropriate for the analysis of human stem and progenitor cell differentiation. Here, using a highly efficient xenotransplantation model, we extensively analyze the origin and the molecular signature of human DCs. Purified human common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) were intravenously transplanted into nonobese diabetic-severe combined immunodeficiency (NOD-scid)/IL2rgamma(null) newborn mice. CMPs and CLPs displayed significant expansion in the xenogeneic host, and human cDC and pDC progeny were isolatable. Strikingly, each human DC subset possessed indistinguishable expression patterns of surface phenotype and gene transcripts regardless of their CMP or CLP origin, even at the genome-wide level. Thus, cDC and pDC normally develop after cells have committed to the myeloid or the lymphoid lineage in human hematopoiesis, while their transcriptional signatures are well preserved irrespective of their lineage origin. We propose that human DCs use unique and flexible developmental programs that cannot be categorized into the conventional myeloid or lymphoid pathway.

Figure 1

Human cord blood Lin⁻hCD34⁺hCD38⁻ HSCs give rise to DC subsets as well as other progenitors. (A) Purification of Lin⁻hCD34⁺hCD38⁻ HSCs from the CD34⁺ cell–enriched cord blood. (B) cDC and pDC progeny in the bone marrow, spleen, and peripheral blood of NOD-scid/IL2rγ^null recipients of human cord blood HSCs. (C) Human HSCs gave rise to myeloid and lymphoid progenitors in the recipients' bone marrow, recapitulating normal human hematopoietic development. (D) The phenotype of purified HSC-derived cDCs and pDCs by FACS. (E) Changes in quantity of IFN mRNA in purified cDCs and pDCs before and after CpG stimulation. Representative data of 2 independent experiments are shown. FSC indicates forward scatter; and BM, bone marrow. Error bars represent the SEM of triplicate cultures.

Figure 2

Human CMPs give rise to cDCs and pDCs in NOD-scid/IL2rγ^null recipients.(A) Purification of human cord blood CMPs based on the expression of lineage antigens hCD34, hCD38, hIL-3Rα, and hCD45RA. (B) Myeloid progeny in the bone marrow 4 weeks after CMP xenotransplantation is shown. Mature hCD13⁺ granulocytes, hCD41a⁺ platelets, and hGPA⁺ erythroid cells were seen in the bone marrow. (C) CMP-derived cDCs and pDCs developed in both the bone marrow and the spleen of CMP recipients. Morphology of purified cDC and pDC progeny is also shown (May-Grünwald-Giemsa staining). See “Analysis of human cell engraftment” for complete image acquisition information. BM indicates bone marrow.

Figure 3

Human CLPs give rise to cDCs and pDCs in NOD-scid/IL2rγ^null recipients. (A) Purification of human cord blood CLPs based on the expression of lineage antigens hCD34, hCD38, and hCD7. (B) The analysis of recipients 4 weeks after CLP xenotransplantation. B- and NK-cell progeny and T-cell progeny were found in the spleen and the thymus, respectively. (C) hCD45⁺ human CLP progeny included cDCs and pDCs in both the bone marrow and the spleen. Morphology of purified cDC and pDC progeny is also shown (May-Grünwald-Giemsa staining). The pDC images presented in 3 separate boxes were taken from a single slide. See “Analysis of human cell engraftment” for complete image acquisition information. BM indicates bone marrow.

Figure 4

Characterization of human CMP- and CLP-derived DC subsets. Flow-cytometric analysis of surface DC–related antigens in human CMP-derived and human CLP-derived cDCs (A) and pDCs (B). The gray area represents each isotype control. Note that the expression patterns of each DC-related antigen are almost identical in cDCs and pDCs, irrespective of their CMP or CLP origin. (C) The relative expression levels of RNAs isolated from cDCs and pDCs in the bone marrow of mice that received CMPs (top) and CLPs (middle) and in normal human bone marrow (bottom). Representative data from 3 independent experiments using real-time PCR analyses are shown. Error bars indicate the SEM of triplicate cultures.

Figure 5

A genome-wide representation of DC subsets of myeloid or lymphoid origin by microarray analysis. (A) Scatter plots of signal intensity of 48 000 different transcripts measured on an Illumina beads chip microarray. Data are presented as CMP- versus CLP-derived pDCs (i), CMP- versus CLP-derived cDCs (ii), CMP-derived cDCs versus pDCs (iii), and CLP-derived cDCs versus pDCs (iv). Representative data of 3 independent experiments are shown. (B) The heat map representation of 218 genes that displayed more than 3-fold differences between cDCs and pDCs of each lineage origin. The expression pattern of these genes is well preserved in each DC subset irrespective of its CMP or CLP origin. Representative data of 3 independent experiments are shown. L and M denote lymphoid and myeloid origin, respectively.

Figure 6

Genes of different expression patterns between each DC subset of myeloid or lymphoid origin. (A) The representative list of 218 genes that displayed significantly different (> 3-fold) expression levels between cDC and pDC subsets. Genes are categorized into groups of genes relevant to apoptosis, CD classification, transcriptional regulation, cytokines, host-pathogen interaction, and signal transduction. All of these functional genes displayed consistent expression patterns irrespective of their lineage origin. (B) List of unknown genes included in the 218 gene subset. Their expression patterns are also preserved in CMP- or CLP-derived DC subsets. Representative data of 3 independent experiments are shown.

Figure 7

A proposed developmental scheme of the DC lineage cells. Human myeloid and lymphoid pathways generate indistinguishable cDC and pDC populations. These DC subsets may use common developmental programs that are independent of the conventional myeloid or lymphoid pathways. It is still unclear whether human cDCs and pDCs develop via the putative common cDC/pDC precursor stage.