CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation

Accepted Manuscript CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation Borim An, Ji-Young Lim, Suji Jeong, Dong-Mi Shin, Eun Young Choi, Chang-Ki Min, Seok-Ho Hong PII:

S0006-291X(17)32187-3

DOI:

10.1016/j.bbrc.2017.11.010

Reference:

YBBRC 38802

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 24 October 2017 Accepted Date: 2 November 2017

Please cite this article as: B. An, J.-Y. Lim, S. Jeong, D.-M. Shin, E.Y. Choi, C.-K. Min, S.-H. Hong, CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.11.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation Borim An1, Ji-Young Lim2, Suji Jeong1, Dong-Mi Shin3, Eun Young Choi4, Chang-Ki Min2,5*, Seok-Ho Hong1* 1

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Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon 24341, South Korea 2 Department of Hematology, Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea 3 College of Human Ecology, Seoul National University, Seoul, Republic of Korea 4 Department of Biomedical Sciences, Seoul National University, College of Medicine, Seoul, Republic of Korea 5 Leukemia Research Institute, The Catholic University of Korea, Seoul, Republic of Korea

Keywords: MDSC, CD1d, T cell suppression, allogeneic HSCT

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*Correspondence: Dr. Seok-Ho Hong Department of Internal Medicine, School of Medicine, Kangwon National University, Kangwondaehakgil-1, Chuncheon, Gangwon-do 24341, Republic of Korea Tel: 82-33-250-7819; Fax: 82-33-244-2367 Email: [email protected]

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Dr. Chang-Ki Min Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea Tel: 82-2-2258-6053; Fax: 82-2-599-3589 Email: [email protected]

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ABSTRACT Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer and various pathological conditions. However, the phenotypic and functional heterogeneity of human MDSCs represents a major hurdle for the development of therapeutic strategies targeting or regulating MDSCs in tumor progression, inflammation, and graft-versus-host disease (GVHD). We previously shown that circulating HLA-DR-CD14+ monocytic MDSCs are a major contributor to clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we identified, using high-throughput screening, a set of surface markers that are strongly expressed in HLADR-CD14+ monocytic MDSCs isolated from the peripheral blood (PB) of patients receiving allo-HSCT. Subsequent experiments showed the consistent dominant expression of CD1d in monocytic MDSCs of allo-HSCT PB in comparison with granulocytic MDSCs. In addition, CD1d-expressing cells isolated from PB of allo-HSCT patients showed the suppressive activity of T cell proliferation and higher expression of MyD88 and IDO compared with CD1d- cells. Our results suggest that CD1d could be a valuable marker for further therapeutic evaluation of human monocytic MDSCs for immune-related diseases, including GVHD.

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1. INTRODUCTION Recently, myeloid-derived suppressor cells (MDSCs) have been characterized as a subset of immune cells from the myeloid lineage that have immunosuppressive roles in vivo as well as in vitro [1]. MDSCs exist at very low frequencies in steady state, but greatly expand in pathological conditions such as cancer, inflammation, infection, autoimmune diseases, and graft-versus-host disease (GVHD) to suppress the excessively activated immune system [2]. Although the suppressive role of MDSCs contributes to protection from inflammation and autoimmunity, it also provides a tumor-friendly microenvironment through the inhibition of T cell functions [3]. Thus, MDSCs have attracted growing interest from the fields of cancer and immune diseases. However, the phenotypic heterogeneity of MDSCs represents a major hurdle to develop therapeutic strategies targeting or regulating them in tumor progression and other immune disorders. In mice, MDSCs are defined as cells co-expressing Gr-1 and CD11b, and can be subdivided into two major populations: monocytic MDSCs (M-MDSCs; CD11b+Gr-1lowLyG6-Ly6Chigh) and granulocytic MDSCs (G-MDSCs; CD11b+Gr-1highLyG6+Ly6Clow) [4]. Based on these cell-surface markers, many studies have identified various other markers to distinguish these two populations from each other. CD49d (also known as integrin-α4) is expressed only in the CD11b+Gr-1low M-MDSC subset, which has more potent immunosuppressive activity compared to the CD11b+ CD49d- cell fraction representing G-MDSCs [5]. F4/80, CD115 (also known as macrophage colony-stimulating factor receptor), and C-C chemokine receptor type 2 are dominantly expressed in M-MDSCs with T cell suppressive activity, which is mediated by the inducible nitric oxide (iNOS) pathway [6,7]. In contrast to murine MDSCs, human MDSCs are not clearly distinguished because of the lack of specific markers. In mononuclear cells of human peripheral blood (PB), G-MDSCs were defined as CD11b+HLADR-/lowCD14-CD15+ and M-MDSCs as CD11b+HLA-DR-/low CD14+CD15- [8]. The CD33 myeloid marker can be used in place of CD11b since very few CD15+ cells are CD11b- [9]. In addition, CD66b can be used instead of CD15 as a marker for G-MDSCs [9]. S100A9 has been recently identified as another marker for human M-MDSCs with T cell inhibitory function [10]. Although some markers have been identified to distinguish M-MDSCs from GMDSCs, no combination of these markers is able exclude dendritic cells, macrophages, and neutrophils. These findings suggest that M- and G-MDSCs are phenotypically and morphologically distinct from each other, and that subpopulations of human MDSCs with stronger immunosuppressive role can exist. Thus, a more detailed phenotypic dissection and functional analysis of MDSCs is needed to develop better therapeutic strategies for cancer and other immune diseases. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important therapeutic modality used to treat hematologic malignancies such as leukemia and lymphoma. However, the use of allo-HSCT is limited by GVHD. Recent studies indicate that circulating M-MDSCs were increased in patients after allo-HSCT [11], and that they reduced GVHD lethality by inhibiting T cell alloresponses in preclinical models [12]. Thus, obtaining an M-MDSC subset with stronger immunosuppressive activity has garnered great attention as a promising way to treat GVHD. Here, we screened cell-surface markers in the PB of allo-HSCT patients and identified a set of markers that are dominantly expressed in M-MDSCs compared to GMDSCs. We propose that these markers could be useful to isolate a specific M-MDSC subset, which might hold therapeutic potential for various immune diseases including GVHD.

ACCEPTED MANUSCRIPT 2. MATERIALS & METHODS

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2.1. Blood samples Peripheral blood was obtained during routine examinations from 40 patients following alloHSCT regardless of donor type, conditioning, underlying disease, and GVHD prophylaxis (Supplemental Table 1). Indications for allo-HSCT were acute myeloid leukemia, acute lymphoid leukemia, and myelodysplastic syndrome. All studies were performed after obtaining the approval of the Institutional Review Board (KC12SISE0585) at the Catholic University of Korea Seoul St. Mary’s Hospital.

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2.2. Cell sorting Human peripheral blood mononuclear cells (PBMNCs) were isolated by differential density gradient separation (Ficoll-Paque, GE Healthcare) within 4 h of the blood draw. MDSCs were isolated using a magnetic-activated cell sorting system (MACS) using HLA-DR, CD14, and CD1d antibodies according to the manufacturer’s instructions (Miltenyi Biotech).

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2.3. Cell-surface marker screening To identify novel markers expressed in the HLA-DR-CD14+ M-MDSC subset, HLA-DRsorted cells were stained with the CD14 antibody. Cells were characterized using the LEGENDScreen human cell screening kit (BioLegend) containing 342 purified monoclonal antibodies, according to the manufacturer’s instructions. Cells were plated into 96-well plates at a density of 3×105 cells per well containing 1% FBS-PBS. Immediately after staining, the analysis was performed using BD FACSCanto II (BD bioscience). The assay was performed using seven donors.

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2.4. Flow cytometry analysis The immunophenotypic characterization was analyzed by flow cytometry as previously described [13]. Briefly, cells were resuspended in 1% FBS-PBS and surface-stained with following fluorochrome-conjugated antibodies for 1 h at 4°C: CD14-APC (BD Bioscience), HLA-DR-FITC, CD1d-PE, CD49a-PE, CD226-PE, CD244-PE, CD317-PE, and CD328-PE (all BioLegend). After being immunostained, the cells were rinsed and stained with 7-amino actinomycin (7AAD) to exclude dead cells. The negative control was a nonspecific IgG from the corresponding cells. Flow cytometric analysis was performed by BD FACSCanto II and FlowJo software (Tree Star).

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2.5. RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from purified MDSCs using the RNeasy mini kit (Qiagen, Limburg, Netherlands) following the manufacturer’s instructions. Reverse transcription for cDNA synthesis was carried out using the TOPscriptTM RT Dry mix (Enzynomics). Real-time quantitative PCR was carried out in triplicate using TOPreal qPCR 2× PreMIX with SYBR Green (Enzynomics) on StepONE plus real-time PCR system (Applied Biosystems). The amplification was performed using the following conditions: 5 min at 95°C, 40 cycles of 15 sec at 95°C, and 1 min at 60°C. Relative gene expression was determined by normalizing to GAPDH. The primers used are listed in Supplemental Table 2. 2.6. T cell suppression assay HLA-DR-CD1d+ and HLA-DR-CD1d- cells purified from PBMNCs from an allo-HSCT patient were subsequently co-cultured with T cells to measure their T cell suppressive

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function. Human T cells were labeled with 2.5 µM CellTrace™ CFSE (Invitrogen) and seeded in 96-well plates at 1×105 cells/well. The T cells were co-cultured with HLA-DRCD1d+ and HLA-DR-CD1d- cells in a 1:1 ratio for 5 days. T cell stimulation was provided by 2 µg/mL of anti-CD3/CD28 (eBioscience) and 5 ng/mL of recombinant human IL-2 (R&D systems). After 5 days of incubation, the cells were stained with anti-CD3-APC-Cy7, antiCD4-PE, and anti-CD8-PE (eBioscience). Proliferation of T cells was analyzed using LSRII (BD Pharmingen) and FlowJo software.

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2.7. Statistical analysis All data were expressed as mean±standard deviation (SD). Comparisons for all experiments were performed using the Student t-test. Significance levels were set at p<0.05.

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3.1. High-content screening identifies a set of cell-surface marker candidates expressed in HLA-DR-CD14+ cells We hypothesized that novel cell-surface markers for isolating the subset of M-MDSCs that possesses immunosuppressive activity exist in the HLA-DR- mononuclear cell fraction of PB from allo-HSCT patients. In order to identify novel surface markers, we collected the PBMNC fraction from the buffy coat fraction from each blood sample and negatively sorted the HLA-DR- cell population using a MACS system. HLA-DR- cells were subsequently stained with APC-conjugated CD14 antibody as previously known as consensus M-MDSC marker and then seeded into plates containing 342 PE-labeled purified monoclonal antibodies. Based on the correlation with CD14 expression in the HLA-DR- subset, the expression patterns of cell-surface molecules were divided into three groups: negative, dim or low, and bright or high (Figure 1A). The initial analysis revealed the presence of 32 positive markers, which showed bright intensity and/or high frequency in the HLA-DR-CD14+ M-MDSC subset (Table 1). As expected, lyoplate analysis confirmed the expression of conventional MMDSC markers including CD11b, CD33, and CD39 on HLA-DR-CD14+ M-MDSCs in highcontent screening. Thus, these three markers were excluded further investigation. We also confirmed that CD40, CD80, and CD83, previously reported as dim or negative markers of M-MDSCs were not detected or very low (Figure 1B). Additionally, CD15 and CD66b, markers for G-MDSCs, were not detected or very low. The initial screening analysis indicated efficient cell isolation and immunostaining of our protocols and suggested that further investigation of the remaining 29 positive surface molecules was needed to identify novel M-MDSC markers (Figure 1C).

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3.2. CD1d and CD226 are novel markers for the human M-MDSC subset To select more potent and specific M-MDSC markers, the remaining 29 surface markers were further investigated based on their phenotypic redundancy with other blood cell types, correlation with CD14 expression, frequency, and the mean fluorescence intensity (MFI). CD49a/f, CD52, CD81, CD84, and CD85h are expressed in monocytes and macrophages, but have phenotypic redundancy with other blood cell types such as erythrocytes, T cells, B cells, natural killer (NK) cells, and dendritic cells. CD11c, CD46, CD53, CD62L, CD69, CD85d/j/k, and CD220 have phenotypic redundancy with other blood cell types including granulocytes. CD49d and CD85j have also phenotypic redundancy with other blood cell types, but were not expressed or identified in granulocytes. Several positive candidates are known as markers for a specific blood cell type. CD24 and CD56 are specific for B cells and NK cells, respectively. CD42b and CD62P are markers for platelets. CD62a/c/e are markers for epithelial cells and granulocytes. CD71, known as a specific marker for erythroid precursors, showed the highest frequency in the HLA-DR- subset, but exhibited lower MFI compared to other markers (Figure 2A and 2B). The remaining six markers, including CD1d, CD49a, CD226, CD244, CD317, and CD328, exhibit phenotypic redundancy with T cells, B cells, and dendritic cells. However, these markers have not been detected or expressed in granulocytes and implicated in inflammatory and immune diseases. In addition, CD226 and CD328 were found to exhibit lower frequency in HLA-DR- subset, but higher MFI in comparison with other markers (Figure 2A and 2B). CD1d and CD49a showed high frequencies and positive correlation with CD14 expression (Figure 2A and 2C). Thus, we selected these six markers as potent novel candidates to facilitate M-MDSC subset discrimination, and further validated their expression.

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We measured the frequencies of these selected markers in HLA-DR-CD14+ cells purified from the PBMNCs of additional HSCT donors (n=15) using flow cytometry. The initial screening showed higher frequencies of CD1d and CD49a in the HLA-DR- subset compared to the frequencies of CD244, CD226, CD317, and CD328. In contrast, additional PBMNC samples indicated that all these markers are expressed at similar levels on the HLA-DRsubset (Figure 3A). However, the correlation with the CD14 M-MDSC marker was consistent with the initial screening analysis (Figure 3B). We next compared transcript levels of these markers between M-MDSCs and G-MDSCs using qRT-PCR. The expression levels of CD1d and CD226 were significantly higher in M-MDSCs compared to G-MDSCs (*p<0.05). CD49a, CD317, and CD328 were slightly higher in M-MDSCs compared to G-MDSCs, but not significantly different. Unexpectedly, the transcript level of CD244 in M-MDSCs was lower than that in G-MDSCs (Figure 3C). These results suggest that the CD1d and CD226 markers could be more specific in identifying M-MDSCs with potent T cell suppressive activity.

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3.3. Gene expression and functional evaluation of CD1d-expressing M-MDSCs We focused on CD1d-expressing M-MDSCs because the functional correlation of CD1d expression with T cell activation and immune diseases including GVHD has been relatively less explored, compared to CD226. First, we compared the expression levels of several genes related to immunosuppressive activity between the HLA-DR-CD1d+ and HLA-DR-CD1dsubsets using qRT-PCR. ARG1, iNOS, and NOX2 transcripts were equivalently detected in the HLA-DR-CD1d+ and HLA-DR-CD1d- fractions. However, transcript levels of myeloid differentiation factor 88 (MyD88) and indoleamine-pyrrole 2,3-dioxygenase (IDO) showed an approximately 3-fold increase in HLA-DR-CD1d+ cells, compared with their expression levels in HLA-DR-CD1d- cells (Figure 4A). To further determine the functional correlation between the higher expression of MyD88 and IDO in HLA-DR-CD1d+ cells and the immunosuppressive activity of these cells, we compared their capacity to suppress T cell proliferation. HLA-DR-CD1d+ and HLA-DR-CD1d- fractions purified from PBMNCs of allHSCT patients were co-cultured with CFSE-labeled naïve T cells stimulated with antiCD3/CD28 antibodies and rhIL-2 at ratio from 1:1. As shown in Figure 4B-D, HLA-DRCD1d+ cells only suppressed the proliferation of CD3+, CD4+, and CD8+ T cells. These results indicate that the gene expression profile and immunosuppressive function of CD1dexpressing M-MDSCs during the development of immune diseases and inflammation deserve further investigation.

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4. DISCUSSION Numerous studies have reported the immunosuppressive roles of MDSCs in various diseases related to immune disorders and inflammation [1,2]. Recent studies showed that M-MDSCs exert more potent suppressive activity on T cells in vitro than do G-MDSCs, through a iNOSmediated pathway [14]. However, the heterogeneous nature of MDSCs limits the clear definition and isolation of the M-MDSC population. Although phenotypes for the identification of murine M-MDSCs have been relatively well defined, human M-MDSCs are not clearly dissected because of the lack of specific markers. Thus, more detailed phenotypic dissection and functional analysis of MDSCs is needed in order to develop better therapeutic strategies to cure immune diseases. In the present study, we identified a set of cell-surface molecules expressed on the HLA-DR-CD14+ M-MDSC subset isolated from PB of alloHSCT patients via high-throughput screening, and further revealed that CD1d could be a novel marker for the isolation of M-MDSCs with T cell suppressive activity. CD1d is a member of the CD1 family (CD1a, b and c) of glycoproteins expressed on the surface of various antigen-presenting cells, and has a wider distribution than do other CD1 subtypes [15]. One regulatory mechanism of the CD1d molecule is to provide glycolipid antigens to NKT cells, a subset of T lymphocytes, and interact with the T cell receptor on their surface. When activated, NKT cells interact with various immune cells by producing a mixture of cytokines, which promote or suppress immune responses in different diseases including autoimmune responses, cancer, and GVHD [16]. MDSCs are rapidly expanded under pathological conditions and acquire immunosuppressive activity, which is mediated by ARG1 and inducible NO synthase. Our previous study demonstrated that MyD88 plays a protective role in developing intestinal GVHD by enhancing the expansion and immunosuppressive functions of MDSCs. After clinical allo-HSCT, T-cell suppressive CD14+HLA-DRlow/neg IDO+ myeloid cells are expanded in PB of patients with GVHD [11]. In addition, it has been reported that NKT cells abolished the immune suppressive activity of MDSCs, which is CD1d- and CD40-dependent [17]. Thus, our data suggest that MyD88expressing CD1d+ M-MDSCs may cooperatively interact with NKT cells, which contributes to protect our body from inflammation and autoimmunity. M-MDSCs are characterized by their immature state as well as their ability to suppress immune responses. There is some evidence to support the notion that CD1d can be a useful marker to identify and isolate the immature monocyte population from PB. Exley M et al. [18] reported that CD1d was detected on CD14+ monocytes freshly isolated from PB. CD1a, b, and c expression was strongly induced in monocytes by stimulation with GM-CSF and IL-4. However, CD1d was not induced under this stimulation, suggesting that CD1d is constitutively expressed in monocytes and not upregulated during maturation. In addition, comparison of transcriptional profiles of CD14highCD16+ and CD14highCD16- monocytes isolated from human PB revealed that CD16+ monocytes represent a more advanced stage of myeloid differentiation with macrophage- and DC-like properties, whereas CD16- monocytes are more closely related to the immature myeloid precursor [19]. More interestingly, CD1d was preferentially expressed in CD16- monocytes compared to CD16+ monocytes, indicating that CD1d could be used as an additional marker to enrich immature M-MDSCs from whole blood. We also identified CD226 as a novel marker for M-MDSCs in allo-HSCT PB. CD226, as an activating receptor on T cells, plays an important role in tumor immunity and the development of GVHD by interacting with the ligands CD155 and CD112 on target cells [20]. Transplantation of allogeneic splenocytes deficient in CD226 resulted in significantly milder GVHD than that resulting from the transplantation of allogeneic wildtype splenocytes in mice [21]. In addition, administration of the anti-CD226 antibody ameliorates GVHD in

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sublethally irradiated mice that received MHC-matched or mismatched bone marrow transplantation, indicating that CD266 could be a therapeutic target for GVHD treatment. Although the role of CD226 in lymphoid lineage cells has been well studied, its expression and function have not been well defined in myeloid lineage cells. One recent study showed that CD226 is expressed on CD11b+ monocytes in mouse spleen and CD14+ monocytes in human PB [22]. Interestingly, CD226 is preferentially expressed in human (CD14+CD16−) and mouse (CX3CR1intCCR2+Ly6Chi) inflammatory monocytes, which play a critical role in host defense against pathogens by promoting their migration ability and cell-to-cell adhesion with CD155-expressing cells [23]. These findings suggest that CD226 can be used as an alternative or combinational marker for CD14 to enrich M-MDSCs with more potent immunosuppressive activity. In summary, we identified CD1d as a novel marker for the isolation of the M-MDSC population with T cell suppressive activity from whole blood. Future studies are needed to further characterize the remaining identified M-MDSC markers and develop an in vitro expansion protocol for M-MDSCs. This will enhance our understanding of immune regulatory mechanisms of M-MDSCs in GVHD and their therapeutic applications for various immune diseases.

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ACKNOWLEDGEMENTS This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (Grant HI16C0047).

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Figure 1. High-throughput screening and identification of a set of cell-surface markers expressed in HLA-DR-CD14+ M-MDSC population of allo-HSCT PB. (A) Schematic showing experimental flow for the screening and identification of cell-surface markers expressed in the HLA-DR-CD14+ M-MDSC population. PBMNCs were collected from PB of seven allo-HSCT patients. Based on their correlation with CD14 expression in HLA-DRsubset, the expression patterns of cell-surface molecules were divided into three groups: negative, dim or low, and bright or high. (B) Confirmation of known positive, dim, and negative markers on M-MDSCs. (C) FACS plot data representing cell-surface markers showing bright intensity and high frequency in the HLA-DR-CD14+ M-MDSC population.

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Figure 2. Further analysis of 29 M-MDSC marker candidates expressed in HLA-DRCD14+ subset. (A) Twenty-nine M-MDSC marker candidates were further investigated based on phenotypic redundancy with other blood cell types, correlation with CD14 expression, frequency, and MFI. Percentage of markers in HLA-DR- cells by flow cytometry. (B) MFI of marker candidates in HLA-DR- cells. (C) Distribution of CD14+ and CD14- cells within the positive population of marker candidates.

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Figure 3. Validation of six selected M-MDSC marker candidates. (A) Percentages of MMDSC marker candidates in HLA-DR- cells by flow cytometry. PBMNCs were collected from the PB of 15 additional allo-HSCT patients for validation. (B) Distribution of CD14+ and CD14- cells within positive population of candidates. (C) Comparison of transcript levels of candidates between M-MDSCs and G-MDSCs by qRT-PCR. All bars indicate the average and standard deviation of three independent experiments. *p<0.05.

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Figure 4. CD1d-expressing M-MDSCs have T cell immunosuppressive activity. (A) qRTPCR analysis for expression levels of genes related with immunosuppressive activity in sorted HLA-DR-CD1d+ and HLA-DR-CD1d- populations. Mean expression normalized against GAPDH. (B-D) T cell suppression mediated by HLA-DR-CD1d+ and HLA-DRCD1d- cells tested at a 1:1 T cell to MDSC ratio. The proliferation of CD3+, CD4+ and CD8+ T cells was measured after co-culturing with HLA-DR-CD1d+ and HLA-DR-CD1d- cells for 5 days. All bars indicate the average and standard deviation of three independent experiments. **p<0.01.

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[17] C. De Santo, M. Salio, S.H. Masri, L.Y. Lee, T. Dong, A.O. Speak, S. Porubsky, S. Booth, N. Veerapen, G.S. Besra, H.J. Grone, F.M. Platt, M. Zambon, V. Cerundolo, Invariant NKT cells reduce the immunosuppressive activity of influenza A virusinduced myeloid-derived suppressor cells in mice and humans, J Clin Invest 118 (2008) 4036-4048. [18] M. Exley, J. Garcia, S.B. Wilson, F. Spada, D. Gerdes, S.M. Tahir, K.T. Patton, R.S. Blumberg, S. Porcelli, A. Chott, S.P. Balk, CD1d structure and regulation on human thymocytes, peripheral blood T cells, B cells and monocytes, Immunology 100 (2000) 37-47. [19] P. Ancuta, K.Y. Liu, V. Misra, V.S. Wacleche, A. Gosselin, X. Zhou, D. Gabuzda, Transcriptional profiling reveals developmental relationship and distinct biological functions of CD16+ and CD16- monocyte subsets, BMC Genomics 10 (2009) 403. [20] C. Bottino, R. Castriconi, D. Pende, P. Rivera, M. Nanni, B. Carnemolla, C. Cantoni, J. Grassi, S. Marcenaro, N. Reymond, M. Vitale, L. Moretta, M. Lopez, A. Moretta, Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule, J Exp Med 198 (2003) 557-567. [21] T. Nabekura, K. Shibuya, E. Takenaka, H. Kai, K. Shibata, Y. Yamashita, K. Harada, S. Tahara-Hanaoka, S. Honda, A. Shibuya, Critical role of DNAX accessory molecule-1 (DNAM-1) in the development of acute graft-versus-host disease in mice, Proc Natl Acad Sci U S A 107 (2010) 18593-18598. [22] C. Rebe, R. Filomenko, M. Raveneau, A. Chevriaux, M. Ishibashi, L. Lagrost, J.L. Junien, P. Gambert, D. Masson, Identification of biological markers of liver X receptor (LXR) activation at the cell surface of human monocytes, PLoS One 7 (2012) e48738. [23] A.V. Vo, E. Takenaka, A. Shibuya, K. Shibuya, Expression of DNAM-1 (CD226) on inflammatory monocytes, Mol Immunol 69 (2016) 70-76.

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Table 1. Screening of cell surface molecules using HLA-DR- cells stained with CD14 Cell surface antigens

Negative

CD1a/b/c, CD3, CD5, CD6, CD8a, CD15, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD31, CD35, CD43, CD44, CD45, CD49c, CD55, CD57, CD58, CD62E, CD64, CD70, CD79b, CD80, CD82, CD85a/g, CD88, CD89, CD90, CD94, CD97, CD99, CD102, CD103, CD108, CD117, CD123, CD127, CD132, CD134, CD135, CD137, CD137L, CD138, CD140a, CD143, CD148, CD150, CD152, CD154, CD156c, CD158a/h, CD158e1, CD158f, CD161, CD162, CD163, CD167a, CD169, CD170, CD172a/g, CD178, CD179b, CD180, CD181, CD183, CD193, CD200, CD201, CD202b, CD203c, CD206, CD207, CD213a2, CD215, CD229, CD235ab, CD253, CD254, CD255, CD257, CD261, CD263, CD266, CD267, CD268, CD271, CD273, CD275, CD278, CD279, CD286, CD290, CD294, CD303, CD304, CD307, CD307d, CD314, CD318, CD324, CD325, CD326, CD334, CD335, CD336, CD337, CD338, CD340, CD351, CD352, CD355, CD360, CLEC9A, 6-opoid receptor, DLL4, EGFR, erbB3/HER3, FcεRIα, FcRL6, Galectin-9, IFN-γ R β

Dim or Low

CD2, CD4, CD7, CD9, CD10, CD11a/b, CD13, CD16, CD26, CD29, CD32, CD34, CD36, CD38, CD40, CD41, CD45RA, CD45RB, CD48, CD50, CD51, CD59, CD61, CD63, CD66b, CD73, CD74, CD83, CD86, CD87, CD93, CD95, CD96, CD100, CD101, CD104, CD105, CD106, CD107a, CD109, CD111, CD112, CD114, CD115, CD116, CD119, CD122, CD124, CD126, CD129, CD131, CD140b, CD141, CD144, CD146, CD155, CD158b/d, CD164, CD165, CD166, CCD172b, CD179a, CD182, CD184, CD195, CD196, CD197, CD200R, CD205, CD209, CD210, CD218a, CD221, CD231, CD243, CD245, CD252, CD258, CD262, CD270, CD274, CD276, CD277, CD282, CD284, CD300e, CD301, CD319, CD344, CD298, CD300e/f, CD317, CD344, CD354, CD357, BTLA, C3AR, C5L2, CCR10, CXCR7, DLL1, DR3, GARP, IL-28RA, CLEC12A

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HLA-DRCD14+

Bright or High

CD1d, CD11b(activated), CD11c, CD14, CD24, CD33, CD39, CD42b, CD46, CD49a, CD49d, CD49e, CD49f, CD52, CD53, CD54, CD56, CD62L, CD62P, CD66a/c/e, CD69, CD71, CD81, CD84, CD85d/h/j/k, CD220, CD226, CD244, CD328

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• High-content screening identified a set of cell-surface marker candidates expressed in HLA-DR-CD14+ M-MDSCs. • CD1d and CD226 are novel markers for the human M-MDSC subset. • CD1d-expressing M-MDSC showed the suppressive activity of T cell proliferation and could be a valuable marker for therapeutic use of human M-MDSCs.