Human 6-sulfo LacNac-positive (slan) cells have been subject to a paradigm debate. They have previously been classified as a distinct dendritic cell (DC) subset. However, evidence has emerged that they may be more related to monocytes than to DCs. To gain deeper insight into the functional specialization of slan cells, we have compared them with both conventional myeloid DC subsets (CD1c and CD141) in human peripheral blood (PB). With the use of genome-wide transcriptional profiling, as well as functional tests, we clearly show that slan cells form a distinct, non-DC-like population. They cluster away from both DC subsets, and their gene-expression profile evidently suggests involvement in distinct inflammatory processes. An extensive transcriptional meta-analysis confirmed the relationship of slan cells with the monocytic compartment rather than with DCs. From a functional perspective, their ability to prime CD4 and CD8 T cells is relatively low. Combined with the finding that "antigen presentation by MHC class II" is at the top of under-represented pathways in slan cells, this points to a minimal role in directing adaptive T cell immunity. Rather, the higher expression levels of complement receptors on their cell surface, together with their high secretion of IL-1β and IL-6, imply a specific role in innate inflammatory processes, which is consistent with their recent identification as non-classical monocytes. This study extends our knowledge on DC/monocyte subset biology under steady-state conditions and contributes to our understanding of their role in immune-mediated diseases and their potential use in immunotherapeutic strategies.

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 (BM) compared to normal bone marrow (NBM) samples. Transcriptional profiling revealed down-regulation of transcripts related to pro-inflammatory pathways in MDS-derived cells as compared to NBM. 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.

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The human bone marrow (BM) gives rise to all distinct blood cell lineages, including CD1c+ (cDC2) and CD141+ (cDC1) myeloid dendritic cells (DC) and monocytes. These cell subsets are also present in peripheral blood (PB) and lymphoid tissues. However, the difference between the BM and PB compartment in terms of differentiation state and immunological role of DC is not yet known. The BM may represent both a site for development as well as a possible effector site and so far, little is known in this light with respect to different DC subsets. Using genome-wide transcriptional profiling we found clear differences between the BM and PB compartment and a location-dependent clustering for cDC2 and cDC1 was demonstrated. DC subsets from BM clustered together and separate from the corresponding subsets from PB, which similarly formed a cluster. In BM, a common proliferating and immature differentiating state was observed for the two DC subsets, whereas DC from the PB showed a more immune-activated mature profile. In contrast, BM-derived slan+ non-classical monocytes were closely related to their PB counterparts and not to DC subsets, implying a homogenous prolife irrespective of anatomical localization. Additional functional tests confirmed these transcriptional findings. DC-like functions were prominently exhibited by PB DC. They surpassed BM DC in maturation capacity, cytokine production, and induction of CD4+ and CD8+ T cell proliferation. This first study on myeloid DC in healthy human BM offers new information on steady state DC biology and could potentially serve as a starting point for further research on these immune cells in healthy conditions as well as in diseases.

Introduction Immune responses play an important role in the pathogenesis and progression of myelodysplastic syndromes (MDS). It has been shown that a consistent immunological signature in MDS comprises an increase in Th17 cells in low risk MDS and an expansion of T regulatory cells (Tregs) in high risk subtypes. Additionally, the presence of high Treg numbers in lower risk stages is an independent prognostic value and is associated with poor survival. The high pro-inflammatory response in low risk MDS is also linked to autoimmune features in these patients. Identification of immune cells that could potentially contribute to the inflammatory environment are of interest to prevent disease progression and, on the other hand, restore excessive immune responses by therapeutic interaction. The exact role of monocytes in this early immune disturbance is yet to be understood. Our preliminary findings showed that MDS-derived monocytes have elevated expression levels of the dendritic cell marker BDCA3/CD141 compared to healthy monocytes. The aim of this study was therefore to investigate the phenotypic and functional characteristics of CD141+ versus CD141- monocytes in MDS patients and compare them with healthy monocytes. Patients and methods Monocytes were gated as CD14hiCD11chiHLA-DR+. The percentage of CD141+ monocytes was determined in 143 MDS bone marrow (BM) and 22 normal bone marrow (NBM) samples, and in the peripheral blood (PB) of 33 MDS patients and 28 normal PB (NPB) samples. Furthermore, CD141 expression was assessed in different disease stages and correlated to several disease characteristics such as the presence of ring sideroblasts (RS), blast percentage and cytogenetic risk group. Additionally, sorted CD141- and CD141+ monocytes of two MDS patients were tested for their functional capacities, including their ability to skew T cells and to up-regulate the co-stimulatory molecules CD80 and CD86 upon stimulation. The production of different cytokines was measured in culture supernatants by using Cytometric Bead Array (CBA). Results The percentage of CD141+ monocytes was highly increased in MDS BM compared to NBM (36% vs 12%, p<0.0001), and, also in MDS PB compared to NPB (24% vs 11%, p=0.012). Additionally, the presence of CD141+ monocytes in PB and BM was positively correlated between the two compartments (r= 0.82, p<0.0001). The increased percentage of BM-derived CD141+ monocytes was more evident in low risk groups (RA, RARS and RCMD) than in high risk groups ([RAEB-I/II], 39% vs 22%, p<0.001), and hence, was associated with low blast counts (<5% vs ≥5% blasts: 40% vs 22%, p=0.004). The presence of RS was also correlated with elevated rates of CD141+ monocytes (50% vs 28%, p=0.002). In addition, MDS patients that showed a very good-to-good cytogenetic risk profile had increased frequencies of CD141+ monocytes compared to patients in poor and very poor risk groups (36% vs 22%, p=0.013). Phenotypic analyses showed elevated expression levels of HLA-DR on CD141+ monocytes compared to total monocytes (1.5 fold, p<0.001). Preliminary functional tests revealed no differences in up-regulation of CD80 and CD86 upon stimulation in CD141+ or CD141- MDS monocytes. However, there was a trend towards higher production of IL-1β, IL-6 and TNF and a reduction in FoxP3+ Treg skewing in cultures of CD141+ monocytes compared to CD141- monocytes. Conclusion In conclusion, CD141+ monocytes were found in much higher frequencies in the BM and PB of MDS patients compared to healthy control BM and PB. Their presence was associated with a low risk stage of the disease, low blast counts, the presence of RS and a very good-to-good cytogenetic risk profile. The first functional analyses point to a high pro-inflammatory state of CD141+ compared to CD141- monocytes. Therefore we hypothesize that these monocytes may contribute to an immune stimulatory environment in low risk MDS patients with potentially a favourable prognostic value. Disclosures No relevant conflicts of interest to declare.

Introduction Immune response plays an important role in the pathogenesis and progression of myelodysplastic syndromes (MDS). It has been shown that a consistent immunological signature in MDS comprises an increase in Th17 cells in low risk MDS and an expansion of Tregs in high risk subtypes. The presence of high Treg numbers in lower risk stages is an independent prognostic value and is associated with poor survival. Whereas the immune response in low risk MDS is pro-inflammatory, high risk MDS is characterized by immunosuppression which could contribute into the expansion of the dysplastic clone and malignant transformation. The exact role of dendritic cells (DC) in inducing this immune dysfunction is yet to be understood. The aim of this comparative study was therefore to investigate the frequency and function of different DC subsets in the bone marrow (BM) and peripheral blood (PB) of MDS patients compared to healthy donors (HD). Patients and methods The enumeration of DC subsets was performed in 110 MDS and in 19 HD bone marrow samples. In peripheral blood 34 MDS and 18 HD samples were investigated. DC were identified by the expression of HLA-DR and the lack of the lineage markers CD14 and CD19. They were further subdivided into CD303+ plasmacytoid DC (pDC), and the CD11c+ myeloid subsets CD1c+ myeloid DC 1 (mDC1), CD141hi myeloid DC 2 (mDC2) and CD16+ 6-Sulfo LacNac (Slan) DC. Furthermore, their ability to upregulate co-stimulatory molecules was assessed by flow cytometry and the secretion of cytokines in response to TLR ligands was analyzed using Cytometric Bead Array (CBA). Their capacity to induce allogeneic T cell proliferation was evaluated in a mixed leukocyte reaction (MLR). Results The frequencies of pDC, mDC1 and mDC2 but not of SlanDC were significantly lower in patients' BM compared to HDs' BM (pDC 0.33% vs 0.72%, p=0.01; mDC1 0.30% vs 0.73%, p<0.001; mDC2 0.01% vs 0.05%, p<0.0001; SlanDC 0.10% vs 0.13%, p=0.20). In PB all DC subsets were significantly lower in patients' samples compared to HDs' samples (pDC 0.15% vs 0.28%, p=0.02; mDC1 0.12% vs 0.61%, p<0.0001; mDC2 0.003% vs 0.045%, p<0.0001; SlanDC 0.19% vs 0.46%, p=0.04). A positive correlation was found between the frequencies of DC subsets in a paired assessment of PB and BM of 30 MDS patients (pDC r=0.77, mDC1 r=0.57, mDC2 r=0.34, SlanDC r=0.60). Of note, in a more advanced disease state DC subsets showed a progressive decline in frequency. To investigate DC function they were isolated or FACS sorted from HD BM (n=3) or patient BM (n=4). Based on their TLR expression profile a combination of LPS and R848 has been used to stimulate DC overnight. In HD upregulation of the co-stimulatory molecules CD80 and CD86 was observed after this stimulation. However, MDS DC stimulated with the same combination of TLR ligands showed a reduced upregulation of CD80 and CD86 (median fluorescence intensity (MFI) CD80: SlanDC 2323 vs 6254, MFI CD86: mDC1 2785 vs 4608; SlanDC 2344 vs 5087). They also produced lower levels of pro-inflammatory cytokines compared to HD DC as measured in culture supernatants (mDC1 TNF [pg/ml] 997 vs 2980, IL-6 [pg/ml] 843 vs 2806, IL-8 [pg/ml] 966 vs 5549 and IL-12p70 [pg/ml] 105 vs 1115). Additionally, after 5 days co-culture of CFSE labeled T cells and DC, mDC1 and SlanDC derived from MDS BM displayed a defective induction of allogeneic CD4+ and CD8+ T cell proliferation compared to HD (mDC1 3.08% vs 10.49% CD4+ T cell proliferation and 4.54% vs 15.07% CD8+ T cell proliferation; SlanDC 1.28% vs 5.23% CD4+ T cell proliferation and 1.50% vs 8.67% CD8+ T cell proliferation). Conclusion Our data clearly point to both a numeric and functional impairment of myeloid DC subsets in BM as well as in PB of patients with MDS. A further decline of DC frequencies in high risk MDS may support the therapeutic targeting of DC in the BM microenvironment of these patients to redress immune dysfunction and possibly prevent further progression to acute myeloid leukemia. Extensive research is needed to further delineate the differences in DC function and immune composition in MDS risk categories. Disclosures Mufti: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.