Cord blood was obtained with written informed consent from the Queensland Cord Blood Bank with approval from the Mater Adult Hospital Human Ethics Committee. CD34⁺ hematopoietic progenitor cells were isolated by density gradient enrichment followed by a positive selection using a CD34⁺ isolation kit (Miltenyi Biotec) as previously described (30). NSG-A2 mice (stock no. 014570) were purchased from Jackson Laboratories. 2–5-day-old NSG-A2 pups received 10 Gy total body irradiation 4 h prior to intrahepatic injection of human CD34⁺ cells. Engraftment of human CD45⁺ cells was confirmed 10–12 weeks later, after which hu mice received 2 s.c. doses of human recombinant huFLT3-L (BioXcell) 4 days apart prior to experimentation. Engrafted mice were injected retro-orbitally with 50 µg poly IC (Invivogen) or 20 µg R848 (Invivogen) alone or in combination and mice were euthanized 2 h later. This study was carried out in accordance with the recommendations of the Australian code for the care and use of animals for scientific purposes (8th Edition). The protocol was approved by the University of Queensland Animal Ethics Committee.

Single cell suspensions of BM, liver, lung, spleen, and peripheral blood from engrafted mice were blocked with rat and mouse serum then labeled with Live Dead^® Aqua (Life Technologies), anti-mouse CD45 PerCP Cy5.5, anti-human CD45 APC Cy7, HLA DR PE Cy7, CD19/20 Pacific blue and either CD141 APC, CD123 PE (all from BioLegend), and CD1c FITC (Abcam) to identify DC, or CD3 Pacific blue, CD8 PE Cy7, CD14 APC (all from BioLegend), and CD4 FITC (BD Biosciences) for other leukocytes (Figure S1 in Supplementary Material). Absolute cell counts were determined by the addition of 5,000 Trucount beads (BD Biosciences) per tube. Data were acquired on a Cyan flow cytometer (Beckman Coulter) and analyzed using Flow Jo software (Tree star, version 8).

Human DCs were enriched from single cell BM suspensions by first labeling with Ab specific for human CD3, CD14, CD19, CD20 (all from Beckman Coulter), CD34 (BD BioSciences), and mouse CD45 (BD BioSciences) and Ter119 (BioLegend) followed by depletion of bound cells using sheep anti-rat IgG Dynabeads (Invitrogen) as previously published (30). Cells were then labeled with Live Dead^® aqua, anti-mouse CD45 PerCP Cy5.5, anti-human CD45 APC Cy7, CD3/CD14/CD19/CD20 Pacific blue, HLA DR PE Cy7, CD123 PE or PerCP Cy5.5, CD1c FITC, and CD141 APC and sorted using a Moflo Astrios (Beckman Coulter) (Figure S2 in Supplementary Material).

Total RNA from purified DC subsets was prepared by resuspending between 1 × 10⁵ and 1 × 10⁶ cells in TRIzol Reagent (Life Technologies) followed by chloroform extraction and isopropanol precipitation. RNA quantity and integrity was measured using the Bioanalyzer RNA Nano chip (Agilent Technologies), with all RNA integrity numbers ranging between 8.4 and 9.7. cDNA was generated from 450 ng RNA and converted to cRNA using an Illumina TotalPrep RNA Amplification Kit (Ambion). A 14 h in vitro transcription was performed. Amplified cRNA (750 ng) for each sample was hybridized onto HumanHT-12 v4 Expression BeadChip Kit (Illumina) according to the manufacturer’s protocol and scanned on an Illumina BeadArray Reader (Illumina). Quality control, normalization, and log 2 transformation of the raw expression data were performed using the Lumi Bioconductor package (31) and integrated into the Stemformatics platform www.stemformatics.org (32) for visualization. Transcripts differentially expressed by CD141⁺ DC and CD1c⁺ DC were identified using ANOVA analysis with p value cut-off threshold of Bonferroni correction p < 0.01 and log(2) 3-fold difference. The data have been deposited in the GEO database, accession # GSE99666, and on Stemformatics http://www.stemformatics.org/datasets/search?ds_id=6612. Publically available data sets and gene signatures for human DC subsets GSE35457 (17) were processed similarly for comparison.

Dendritic cell subsets purified from hu mice were exposed to TLR adjuvants for 18 h in vitro, or cultured in media alone for 18 h. Costimulatory molecule expression on the cell surface was quantified by flow cytometry. Culture supernatants and serum samples from the mice were analyzed for human cytokines using a LEGENDplex™ flow cytometry bead array kit (Biolegend). Human IFN-λ was measured by ELISA (R&D Systems).

HLA-A2⁺ DC subsets isolated from hu mice were pulsed with the HLA-A2-restricted MART-1 peptide, ELAGIGILTV (1 µM) in the presence of poly IC and R848 for 2 h at 37°C. Naïve CD8⁺ T cells from autologous cord blood mononuclear cells were purified by negative selection using CD8⁺ T cell isolation kit (Miltenyi Biotec). Peptide-pulsed DCs were cultured with naïve CD8⁺ T cells in the presence of IL-2 (Hoffman-LaRoche) and T cell growth factor for 21 days. MART-1-specific CD8⁺ T cells were quantitated by flow cytometry using a MART-1/HLA-A2-specific pentamer-APC (ProImmune), Live Dead^® aqua, and anti-human CD19-Pacific blue, CD14-Pacific blue, CD3-PE, and CD8-FITC antibodies (BioLegend). Samples were acquired on a LSR Fortessa flow cytometer (BD Biosciences) and analyzed using Flow Jo software (Tree star, version 8). For polyfunctional T cell assays, the CD8⁺ T cell cultures were restimulated for 6 h in the presence of absence of MART-1 peptide (1 µM) with brefeldin A, GolgiStop, and anti-CD107a-FITC (BD Biosciences). Cells were then washed and labeled with anti-CD4 BV711, anti-CD8 APC Cy7, and Live dead^® followed by permeabilization (Cytofix/Cytoperm; BD Biosciences) and stained with anti-TNFα APC, anti-IFNγ PE, and anti-IL-2 Alexa700. Samples were acquired on a LSR Fortessa cytometer (BD Biosciences) and analyzed using Flow Jo software (Tree star, version 8). Boolean gating was performed to measure the frequency of each response based on all possible functional combinations of IFNγ, TNFα, IL-2, and CD107a. The data were analyzed and graphed using Simplified Presentation of Incredibly Complex Evaluations (SPICE) software, version 5.3.

Robust engraftment of human CD45⁺ cells was observed 10–14 weeks after reconstitution of 2–5-day-old NSG-A2 pups with human cord blood-derived CD34⁺ HSC (Figure 1A; Figure S1 in Supplementary Material). Human CD45⁺ cells were most prevalent in the spleen and liver, where they comprised 57.34% + 17.97 (mean + SD, n = 9) and 67% + 21.45 (mean + SD, n = 5) of mononuclear cells, respectively. Human CD45⁺ cells comprised 39.88% + 15.66 (mean + SD, n = 5) of mononuclear cells in the BM and could also be detected in lower proportions in the lungs and blood (Figure 1B). Human CD141⁺ and CD1c⁺ DC subsets were found in all organs examined, with the largest numbers residing in the BM compartment (Figure 1C). Human B cells, CD14⁺ monocytes, plasmacytoid DC, and CD4⁺ and CD8⁺ T cells also developed in this model (Figure S1 in Supplementary Material). Thus, NSG-A2 mice support robust, multi-lineage development of human leukocytes, including CD141⁺ and CD1c⁺ DC.

We compared transcriptomes of BM-derived CD141⁺ and CD1c⁺ DC from hu mice with equivalents from human blood (17). We identified 316 genes that were significantly differentially expressed (log-3 fold change, adjusted p < 0.01) between CD141⁺ and CD1c⁺ DC regardless of tissue source (Table S1 in Supplementary Material). Consistent with known DC functions, gene set enrichment analysis of these subsets ranked C-type lectins, endosomal TLR pathways, and the AIM2 inflammasome as most discriminating (Table S2 in Supplementary Material). Hierarchical clustering analysis of the differentially expressed genes revealed clustering of hu mouse CD141⁺ DC with human blood CD141⁺ DC, and hu mouse CD1c⁺ DC with human blood CD1c⁺ DC (Figure 2A). In an unsupervised clustering approach, the first component of a principal component analysis was concordant with batch, tissue, and species source (hu mice versus human blood). However, clustering of primary human CD141⁺ DC with hu mouse CD141⁺ DC, and CD1c⁺ DC across the two datasets with human peripheral blood monocytes was evident between the first and second principal components (Figure S3 in Supplementary Material).

Amongst the 156 genes preferentially expressed by hu mouse CD141⁺ DC and blood CD141⁺ DC were genes previously associated with this subset including CLEC9A, CADM1, ID2, BATF3, RAB7B, AIM2, BTLA, SEPT3, CLNK, and GSAM (Figure 2B; Table S1 in Supplementary Material). IRF8, TLR3, and XCR1 were also more highly expressed by CD141⁺ DC (Figure 2B; Figure S4 in Supplementary Material). Genes known to be expressed by the CD1c⁺ DC lineage, including IRF4, SIRPA, CLEC10A, FCGR2B, FCER1A, TLR5, and TLR7 were amongst the 160 top ranked differentially expressed genes in hu mouse and human blood CD1c⁺ DC (Figure 2B; Table S1 in Supplementary Material). CD1c⁺ blood DCs have recently been subdivided into two subsets defined as “non-inflammatory” or “CD1c A”, defined by expression of FCER1A, CLEC10A, and FCGR2B; and “inflammatory” or “CD1c B” defined by expression of CD36 and CD163 along with inflammatory genes including CD14, S100A9, S100A8 (5). Hu mouse CD1c⁺ DC expressed much higher levels of the CD1c A non-inflammatory markers than hu mouse CD141⁺ DC, but also expressed the inflammatory markers CD163, CD14, S100A9, and S100A8, which were further upregulated after activation (Figure S5 in Supplementary Material). These data indicate that the hu mouse CD1c⁺ DC isolated here likely contain both inflammatory and non-inflammatory CD1c⁺ DC subsets. Because CD1c⁺ DC have known overlapping features with monocytes (20), we used differential expression analysis to identify genes that CD1c⁺ DC and monocytes co-express. More than 820 differentially expressed genes were identified by comparing hu mouse CD1c⁺ DC, blood CD1c⁺ DC, and blood monocyte subsets with CD141⁺ DC and pDC (log-3 fold change, adjusted p < 0.01, and Figure S6 in Supplementary Material). Genes shared between monocytes and CD1c⁺ DC included CSF1R, SIRPA, IL1B, NLRP3, CASP1, FCGR2A, and FCGR2B.

TLR expression and maturation by human DC are well characterized in vitro but their responses in vivo are not. The TLR3 ligand, poly I:C, and the TLR7/8 ligand, R848, have been used as adjuvants to activate human DC alone or in combination (21, 28, 33, 34). Because cytokines can be detected in mouse serum within 1 h of adjuvant injection (35), we chose a 2 h time point to investigate the early changes in gene expression associated with activation of BM derived CD141⁺ DC and CD1c⁺ DC in vivo. A one-way AVONA analysis (adjusted p < 0.05) revealed differential expression of 408 genes as a result of activation. These genes were involved in immune signaling pathways including Tnfr2 signaling, IFNα/β signaling, Tnfr1 signaling (29% of pathway genes represented, p = 9.05E-04), canonical NF-κB pathway (17% of pathway genes represented, p = 0.027), RIG-I/MDA5, and TLR signaling (Figures 3A,B; Table S3 in Supplementary Material). Twenty five of these genes belong to a core set of NK-κB or IFN-stimulated genes (ISG) associated with DC maturation in both mouse and human irrespective of stimuli (Table S4 in Supplementary Material) (36). Most of the genes differentially expressed after activation were upregulated to similar degree by CD141⁺ DC and CD1c⁺ DC in response to all stimulatory conditions. These included TNFAIP3, TRAF1, IFIT1, ITIF2, ISG15, and OASL (Figure 3A). A small subset of genes were significantly upregulated by both CD141⁺ DC and CD1c⁺ DC in response to R848 alone or combined with poly I:C but were only marginally upregulated by poly I:C alone. These included immunoregulatory molecules LTA, NFIL3, CD274, and IGFB4 (Figure 3B). Conversely, no genes were found to be significantly upregulated on both subsets activated with poly I:C that were not also upregulated by R848.

Poly I:C and R848 alone or combined induced expression of costimulatory molecule genes CD40, CD80, and CD83 to similar levels by both CD141⁺ DC and CD1c⁺ DC (Figure 4A). Gene expression correlated with increased protein expression of CD40, CD80, and CD83 on the surface of purified CD141⁺ DC and CD1c⁺ DC after in vitro activation (Figure 4B). However, cell surface expression of CD80 was significantly higher on activated CD141⁺ DC compared to activated CD1c⁺ DC, and while CD83 expression was also higher on activated CD141⁺ DC, this did not reach statistical significance. Similarly, chemokine genes CXCL9 and CXCL10 were upregulated by both DC subsets following activation but only CXCL10 reached statistical significance (Figure 5A). Upregulation of CXCL9 and CXCL10 correlated with detectable levels of these chemokines in the serum of hu mice 2 h after activation (Figure 5B). In vitro, purified CD141⁺ DC were the main producers of CXCL9 while both subsets produced high levels of CXCL10 (Figure 5C). Collectively, these data highlight both common and distinct pathways of CD141⁺ DC and CD1c⁺ DC that are activated by R848, poly I:C, or the combination.

The mouse CD11b⁺ equivalents of CD1c⁺ DC promote Th17 responses (20). Consistent with this, we found genes encoding the Th17-promoting cytokines IL1B and IL6 were upregulated by both BM derived DC subsets after activation, but levels expressed by activated CD1c⁺ DC were fourfold and eightfold higher than similarly activated CD141⁺ DC, respectively (Figure 6A). Importantly, expression of IL23A was 19-fold higher in CD1c⁺ DC activated with R848 alone or combined with poly I:C compared to similarly activated CD141⁺ DC. IL-1β, IL-6, and IL-23 were detectable in the serum of hu mice and although purified hu mouse CD1c⁺ DC produced higher amounts of these cytokines in vitro compared to CD141⁺ DC, the levels did not reach statistical significance (Figures 6B,C). TNF and IL-12A were similarly upregulated by in vivo activated CD141⁺ DC and CD1c⁺ DC with cytokines detectable in the serum. Higher levels of TNF and IL-12p70 were produced by in vitro cultured CD1c⁺ DC compared with CD141⁺ DC, although this did not reach statistical significance (Figure 6C).

CD141⁺ DC have previously been shown to produce IFNα after poly I:C activation in vivo (28). Here, we found genes for IFNα subtypes IFNA2, IFNA7, and IFNA21 were upregulated to a similar degree by both subsets, most notably in response to R848 alone or combined with poly I:C, while genes encoding most other IFNα subtypes were below detectable levels in both BM-derived DC subsets (Figure 7A). Although large amounts of IFNα were detectable in the serum of mice treated with R848 and R848 + poly I:C, no IFNα was detected in the serum of mice treated with poly I:C alone (Figure 7B). Moreover, IFNα was not detectable in the supernatants of purified CD141⁺ or CD1c⁺ DC activated in vitro (data not shown). IFNB1 was upregulated by both subsets in response to all stimuli. CD141⁺ DC are known to produce large amounts of IFNλ in response to poly I:C (14) and consistent with this, IFNλ genes IL28A, IL28B, and IL29 were expressed 13-, 17-, and 8-fold higher by poly I:C + R848-activated CD141⁺ DC compared to similarly activated CD1c⁺ DC (Figure 7A). This correlated with detection of IFNλ in the serum and in the supernatants of purified CD141⁺ DC activated in vitro (Figure 7C). These data confirm that CD141⁺ DC are major producers of IFNλ in vivo.

MYCL is required for optimal CD8⁺ T cell priming by mouse DC (37) and was expressed at 10-fold higher levels by CD141⁺ DC activated with either poly I:C or R848 compared to similarly activated CD1c⁺ DC (Figure 8A). This suggested an enhanced capacity for activated CD141⁺ DC to prime naïve CD8⁺ T cells that may be independent of their enhanced capacity for cross-presentation. To address the potential for enhanced priming ability by activated CD141⁺ DC, we purified human HLA-A2⁺ DC subsets from hu mice and pulsed them with the HLA-A2-restricted peptide from the melanoma Ag MART-1 after activation with combined poly I:C + R848. Peptide pulsed, activated CD141⁺ DC and CD1c⁺ DC were used to prime autologous, naïve cord blood CD8⁺ T cells. CD141⁺ DC primed a significantly higher percentage of MART-1-specific CD8⁺ T cells compared to CD1c⁺ DC (Figure 8B). However, there was no difference in the polyfunctional capacity of the T cells by either subset, as measured by production of effector molecules IFNγ, TNF, IL-2, and CD107a (Figure 8C).