Reduced frequencies and functional impairment of dendritic cell subsets and non-classical monocytes in myelodysplastic syndromes

Abstract

In myelodysplastic syndromes (MDS) the immune system is involved in pathogenesis as well as in disease progression. Dendritic cells (DC) are key players of the immune system by serving as regulators of immune responses. Their function has been scarcely studied in MDS and most of the reported studies didn't investigate naturally occurring DC subsets. Therefore, we here examined the frequency and function of DC subsets and slan+ non-classical monocytes in various MDS risk groups. Frequencies of DC as well as of slan+ monocytes were decreased in MDS bone marrow compared to normal bone marrow samples. Transcriptional profiling revealed down-regulation of transcripts related to pro-inflammatory pathways in MDS-derived cells as compared to normal bone marrow. Additionally, their capacity to induce T-cell proliferation was impaired. Multidimensional mass cytometry showed that whereas healthy donor-derived slan+ monocytes supported Th1/Th17/Treg differentiation/expansion their MDS-derived counterparts also mediated substantial Th2 expansion. Our findings point to a role for an impaired ability of DC subsets to adequately respond to cellular stress and DNA damage in the immune escape and progression of MDS. As such, it paves the way toward potential novel immunotherapeutic interventions.

Figure 1.

Cell subset enumeration in myelodysplastic syndrome- and healthy-derived bone marrow and peripheral blood. (A) Gating strategy of dendritic cells (DC) and slan+ non-classical monocytes in normal bone marrow (NBM) and myelodysplastic syndromes (MDS)-derived BM. After debris and doublet exclusion, CD45⁺ mononuclear cells were gated. Then plasmacytoid DC (pDC), myeloid DC (cDC1 and cDC2) and slan+ monocytes were identified based on the expression of CD141^high, CD1c and M-DC8/CD16, respectively. (B) Frequencies of different cell subsets in normal bone marrow (NBM) compared to MDS BM. In total 30 NBM samples and 187 MDS BM samples were used. Percentages were calculated from the mononuclear cell fraction. Mean frequencies ± standard error of the mean (SEM) are given (NBM vs. MDS BM: pDC 0.76% SEM ± 0.09 vs. 0.91% SEM ± 0.11, cDC1 0.048% SEM ± 0.006 vs. 0.030% SEM ± 0.003, cDC2 0.67% SEM ± 0.05 vs. 0.54% SEM ± 0.04 and slan+ 0.36% SEM ± 0.07 vs. 0.24% SEM ± 0.02). (C) Cell frequencies in different classification groups according to the 2016 World Health Organization (WHO) classification. Patients having a higher blast count-related 2016 WHO classification (EB-1/EB-2) show lower percentages of DC and slan+ monocytes compared to NBM and lower risk groups (SLD/MLD/RS-SLD/RS-MLD). NBM (n=30) vs. (RS-)SLD/MLD (n=115) vs. EB-1/EB-2 (n=48): pDC 0.76% vs. 1.11% vs. 0.63%, cDC1 0.048% vs. 0.038% vs. 0.015%, cDC2 0.67% vs. 0.59% vs. 0.44%, slan+ 0.36% vs. 0.24% vs. 0.23%. (D) Cell frequencies in different risk groups within the International Prognostic Scoring System (IPSS). The percentages of myeloid DC subsets decrease gradually in higher risk groups. NBM (n=30) vs. low risk (n=49) vs. intermediate-1 (n=71) vs. intermediate-2 (n=21) vs. high risk (n=5): cDC1 0.048% vs. 0.035% vs. 0.031% vs. 0.010% vs. 0.005%, cDC2 0.67% vs. 0.57% vs. 0.52% vs. 0.42% vs. 0.26%. (E) Cell frequencies in different risk groups within the IPSS-R. Again, differences between subgroups are mainly seen in DC subsets. Higher risk groups show lower percentages of DC compared to NBM and lower risk groups. NBM (n=30) vs. very low/low risk (n=77) vs. intermediate risk (n=32) vs. high/very high risk (n=27): cDC1 0.048% vs. 0.038% vs. 0.019% vs. 0.013%, cDC2 0.67% vs. 0.62% vs. 0.41% vs. 0.35%. (F) Correlation of cell frequencies in MDS-derived peripheral blood (PB) and BM samples. In total, 26 paired MDS samples were included. The non-parametric Spearman’s correlation test was used to find significant correlations between frequencies in PB and BM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. EB: excess blasts; MLD: multilineage dysplasia; RS-MLD: ring sideroblasts with multilineage dysplasia; RS-SLD: ring sideroblasts with single lineage dysplasia; SLD: single lineage dysplasia.

Figure 2.

Clonal involvement of dendritic cells subsets and slan+ monocytes. Fluorescence in situ hybridization (FISH) analysis of sorted cells, including B cells and CD34⁺ blast cells, with a known cytogenetic aberrancy. In three tested cases (monosomy 7, del 5q and trisomy 8), isolated cDC2 and CD34⁺ blast cells were highly involved in the dysplastic clone, whereas B cells were not involved. Slan+ monocytes showed clonal involvement in del5q. Interphase FISH on whole bone marrow samples showed both an aberrant and a normal cell line. A representative FISH analysis is shown in which interphase cells are hybridized with the chromosome 5q probe displayed in red and 5p probe displayed in green (LSI EGR1(5q31)/D5S23,D5S721(5p15.2) Dual Colour Probe Set). Loss of 5q is seen in CD34⁺ blasts, cDC2 and slan+ monocytes (2G1R), but not in B cells (2G2R).

Figure 3.

Transcriptomic comparison between healthy donor- and myelodysplastic syndrome-derived cell subsets. Three healthy donor (HD)-derived samples and four myelodysplastic syndromes (MDS)-derived samples were used for the isolation of cDC2 and slan+ monocytes and subsequent microarray analysis. (A) Hierarchical clustering, based on differentially expressed genes, of replicate samples for cDC2 and slan+ monocytes. Heatmap visualization is used to show transcript clustering for the two different conditions (HD vs. MDS). (B) Volcano plots showing overand under-expressed genes in red and green, respectively, in HD compared to MDS. A cut-off of -2.5 / 2.5 for foldchange and a P-value < 0.05 were used to show results. (C) Pathway analyses for transcripts that are under-expressed in MDS compared to HD for cDC2 and slan+ monocytes. Coding differentially expressed genes (774 genes for cDC2 and 987 genes for slan+ monocytes) were selected and imported into the STRING v10.5 bioinformatics tool. Six enriched biological processes with lowest false discovery rate (FDR) are shown for cDC2 and slan+ monocytes (in total, eight enriched pathways were found for cDC2 and 383 for slan+ monocytes). (D and E) Gene set enrichment analysis for HD- and MDS-derived cDC2 (D) and slan+ monocytes (E). For both subsets five gene sets were enriched in HD. An enrichment plot is displayed for each subset. Heatmaps show the core enriched genes (67 for cDC2 and the top 75 of 280 for slan+ monocytes) with interesting genes highlighted by black stars. GO: gene ontology; NES: normalized enrichment score; Nom P-value, nominal P-value.

Figure 4.

Leading-Edge Analysis. (A) Leading-Edge Analysis using five gene sets for both cell subsets. The tables show total number of genes that are present in a specified gene set and the number and percentage of genes that were considered to form the leading-edge subset of that gene set. (B) A set-to-set analysis for cDC2 and slan+ monocytes. Overlap in leading-edge genes between gene sets are displayed using a color intensity graph. A dark green cell indicates that sets have the same leading-edge genes. (C) Heatmap of the leading-edge subset for cDC2 and slan+ monocytes. Genes displayed are present in the leading-edge subset of all five gene sets. The heatmaps show relative expression levels per gene between healthy donor (HD) and myelodysplastic syndrome (MDS) samples. FDR: false discovery rate; GO: gene ontology; NES: normalized enrichment score; Nom P-value: nominal P-value.

Figure 5.

Functional capacities of dendritic cell subsets and slan+ monocytes. (A) Maturation capacity of cDC2 and slan+ monocytes in myelodysplastic syndrome (MDS) bone marrow (BM) upon toll-like receptor (TLR)-stimulation. Expression levels of CD80, CD86 and HLA-DR were assessed by flow cytometry at baseline (T=0) and after overnight stimulation with LPS + R848 (+). Mean fluorescence intensity (MFI) values of these three markers were measured. Median values of 4-7 experiments are shown. (B) Up-regulation of CD80, CD86 and HLA-DR after overnight TLR-stimulation in normal bone marrow (NBM)- (n=4) and MDS-derived (n=4-7) cDC2 and slan+ monocytes. Median values are shown. (C) Cytokine secretion assay. Culture supernatants of healthy (n=4) and MDS (n=10) BM-derived unstimulated and stimulated cDC2 and slan+ monocytes were analyzed for the presence of different cytokines by cytometric bead array. Median values are shown. (D) Allogeneic mixed leukocyte reaction (MLR). Peripheral blood lymphocytes (PBL) were labeled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with healthy (n=2-4) or MDS-derived (n=1-5) stimulated cDC1, cDC2 or slan+ monocytes. The percentage of CFSE-diluted T cells was determined by flow cytometry. Median values of different experiments are shown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. pg: picogram.

Figure 6.

Mass cytometry of T cells co-cultured in the presence of healty donor- or myelodysplastic syndrome-derived slan+ monocytes. Healthy donor (HD)-derived CD4+ T cells were co-cultured in the presence of slan+ non-classical monocytes from healthy donors (n=2) or from myelodysplastic syndrome (MDS) patients (n=2). T cells at the start of the experiment (named “day 0”) as well as T cells co-cultured for 5 days with slan+ non-classical monocytes were stained with a panel consisting of surface markers and intracellular markers, and markers for transcription factors and cytokines and analysed using mass cytometry (CyTOF). First, viable T cells were identified for each experiment. Then the FlowSOM algorithm was used to identify 15 metaclusters containing cells that express the same set of markers. (A) T cells are visualized using viSNE plots. The expression of a selection of markers are shown in the viSNE plots for cultures containing HD- or MDS-derived slan+ monocytes at day 0 and day 5. T-cell subsets were identified based on the expression of IFN-, Tbet, IL-4, GATA3, IL-17, CD25, CD127, IL-10 and FoxP3 (Th1 were considered to be IFN-and Tbet⁺, IL-17 and GATA3^–; Th2 were GATA3⁺ or IL-4⁺; Th17 were IL-17⁺; Tregs were CD127- and FoxP3⁺CD25⁺). (B) FlowSOM-identified metaclusters were laid over day 0 and day 5 viSNE maps. Percentages of identified T-cell subsets at the start of the experiment and at day 5 are shown. Compared to day 0, HD-derived slan+ non-classical monocytes mainly induced pro-inflammatory T cells (Th1 and Th17), as well as collateral Tregs. In contrast, T cells cultured in the presence of slan+ non-classical monocytes from MDS patients showed Th1, and above all, Th2 skewing. In HD-derived cultures Th2 cells disappeared at day 5.