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N. van Leeuwen-Kerkhoff et al.
might contribute to the pathogenesis of these malignan- cies and could be an important target for therapy.14-16 In MDS different types of immune cells are believed to play a role in pathophysiology.17,18 Low-risk disease is often characterized by an activated immune system in which pro-inflammatory cells are numerically increased.19,20 In contrast, it has been demonstrated in high-risk disease that immunosuppressive cell types, e.g., Tregs and myeloid derived suppressor cells (MDSC), are expanded and eventually facilitate immune escape and disease pro- gression.20-23 Limited data is available on the role of DC in different MDS risk groups. Thus far, the focus of most studies has been on the frequencies and function of either in vitro generated monocyte-derived DC (MoDC) or total DC rather than functionally distinct DC subsets.24-27
In this study, we have investigated the frequencies of pDC and myeloid subsets (cDC and slan+ non-classical monocytes) in the BM and peripheral blood (PB) of differ- ent MDS risk group patients (i.e., low- and high risk based on the International Prognostic Scoring System [IPSS] and Revised IPSS [IPSS-R] or using the 2016 World Health Organization [WHO] classification) and compared them to normal BM (NBM) samples. Furthermore, we performed fluorescence in situ hybridization (FISH) analysis to demonstrate clonal involvement. A genome wide transcriptional analysis was carried out to find dif- ferences between healthy donor (HD) and MDS-derived subsets. In functional assays, their maturation and cytokine secreting capacity as well as their ability to induce T-cell proliferation was assessed. Their reduced frequencies and a selective functional impairment, related to danger and tissue damage responsiveness, provide clues as to the role of these myeloid APC subsets in MDS progression.
Methods
Patient and control samples
In this study, 30 NBM samples and 187 BM and 26 PB samples of newly diagnosed MDS patients were used. Risk scores accord- ing to IPSS28 and the IPSS-R29, were available for 150 and 136 patients, respectively. The 2016 WHO classification was available for 163 patients (details are given in the Online Supplementary Appendix and the Online Supplementary Table S1). NBM samples were obtained after written informed consent from patients who were undergoing cardiac surgery and were considered hematolog- ically healthy (i.e., no cytopenia, normal morphology and normal flow cytometric profile). The study was approved by the local Institutional Review Board and was in accordance with the decla- ration of Helsinki.
Enumeration of antigen presenting cells subsets and fluorescence in situ hybridization
PB and BM cells were analyzed on a flow cytometer (FACSCantoTM, BD Biosciences) after incubation with a panel of monoclonal antibodies (see the Online Supplementary Appendix for details). After debris and doublet exclusion, cell subtypes were identified in the CD45+ compartment. CD141, CD1c, CD303 and M-DC8/CD16 were used for the identification of cDC1, cDC2, pDC and slan+ monocytes, respectively (Figure 1A). Frequencies of all populations were calculated as percent- ages of CD45+ mononuclear cells.
Three MDS samples with a known cytogenetic aberrancy were used for the isolation of cDC2 and slan+ monocytes and
subsequent FISH analysis (details are given in the Online Supplementary Appendix).
Functional assays and multidimensional mass cytometry
The maturation capacity, the secretion of cytokines and the abil- ity to induce T-cell proliferation was tested for MDS BM- and NBM-derived cDC2 and slan+ non-classical monocytes. See the Online Supplementary Table S1 for clinical data. A multi-parameter deep-phenotyping strategy, known as cytometry by time-of-flight (CyTOF), was used for T cells cultured in the presence of MDS- derived or healthy PB-derived slan+ monocytes. DC subsets could not be included in this experiment because of low cell numbers. See the Online Supplementary Appendix file for technical details.
Microarray transcriptional analysis
RNA was isolated from MDS BM- and NBM-derived cDC2 and slan+ monocytes (5.000-67.000 cells) and amplified using the Ovation Pico WTA System V2 (NuGen, San Carlos, CA) as previously described.11 RNA was labeled with the Encore Biotin Module Kit (NuGEN) and 5 mg of cDNA from each sample was hybridized to Human Transcriptome Arrays 2.0 microarrays (Affymetrix) and signals were scanned by Affymetrix GeneChip Scanner 3000 7G. See the Online Supplementary Appendix for details on data analysis. The microarray data have been deposit- ed in the GEO public database under the accession number: GSE161058.
Statistical analysis
Graphpad Prism 6 software (San Diego, USA) was used for flow cytometry and functional data analysis and graphic display. For two-group comparisons, differences were assessed by apply- ing a non-parametric Mann-Whitney U test. Multi-group com- parisons were analyzed with a Kruskal-Wallis with Dunn’s mul- tiple comparisons test. The non-parametric Spearman’s correla- tion test was used for correlations. A P-value of <0.05 was con- sidered significant.
Results
Dendritic cell frequencies are reduced in the bone marrow of MDS patients
Frequencies of different DC subsets and slan+ mono- cytes were analyzed in BM samples of 187 newly diag- nosed myelodysplastic syndrome patients (detailed in Table 1) and compared to 30 NBM samples. An eight- colour flow cytometry panel was used for the detection of CD303+ pDC, CD141hi cDC1, CD1c+ cDC2 and M-DC8+/CD16+ non-classical monocytes (Figure 1A). Except for pDC, all subsets showed significantly lower frequencies in MDS-derived BM compared to NBM (Figure 1B; NBM vs. MDS BM: cDC1 0.048% vs. 0.030%, cDC2 0.67% vs. 0.54% and slan+ 0.36% vs. 0.24%). pDC rates were increased in MDS BM (NBM, 0.76%; MDS BM, 0.91%). This was mainly observed in cases that were associated with low blast counts (Figure 1C; NBM, 0.76%; (RS-)SLD/MLD, 1.11%; EB-1/EB-2, 0.63%). For cDC1 and cDC2, frequencies gradually decreased in classification groups associated with higher risk MDS (Figure 1C; NBM, 0.048%; (RS-)SLD/MLD, 0.038%; EB-1/EB-2, 0.015% and NBM, 0.67%; (RS-)SLD/MLD, 0.59%; EB-1/EB-2, 0.44%, respectively). Also, for slan+ monocytes lowest frequen- cies were found in the EB-1/EB-2 classification group (NBM, 0.36%; (RS-)SLD/MLD, 0.24%; EB-1/EB-2,
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