Inhibition of Immune Synapse by Altered Dendritic Cell Actin Distribution: A New Pathway of Mesenchymal Stem Cell Immune Regulation This information is current as of September 27, 2021. Alessandra Aldinucci, Lisa Rizzetto, Laura Pieri, Daniele Nosi, Paolo Romagnoli, Tiziana Biagioli, Benedetta Mazzanti, Riccardo Saccardi, Luca Beltrame, Luca Massacesi, Duccio Cavalieri and Clara Ballerini

J Immunol 2010; 185:5102-5110; Prepublished online 1 Downloaded from October 2010; doi: 10.4049/jimmunol.1001332 http://www.jimmunol.org/content/185/9/5102 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Inhibition of Immune Synapse by Altered Dendritic Cell Actin Distribution: A New Pathway of Mesenchymal Stem Cell Immune Regulation

Alessandra Aldinucci,* Lisa Rizzetto,† Laura Pieri,‡ Daniele Nosi,‡ Paolo Romagnoli,‡ Tiziana Biagioli,* Benedetta Mazzanti,x Riccardo Saccardi,x Luca Beltrame,† Luca Massacesi,* Duccio Cavalieri,† and Clara Ballerini*

Immune synapse formation between dendritic cells (DCs) and T cells is one of the key events in immune reaction. In immunogenic synapses, the presence of fully mature DCs is mandatory; consequently, the modulation of DC maturation may promote tolerance and represents a valuable therapeutic approach in autoimmune diseases. In the field of cell therapy, bone marrow mesenchymal stem cells (MSCs) have been extensively studied for their immunoregulatory properties, such as inhibiting DC immunogenicity Downloaded from during in vitro differentiation and ameliorating in vivo models of autoimmune diseases (e.g., experimental allergic encephalomyelitis). MSCs seem to play different roles with regard to DCs, depending on cell concentration, mechanism of stimulation, and accompanying immune cells. The aim of this work was to elucidate the immunogenic effects of MSC/DC interactions during DC activation (LPS stimulation or Ag loading). Human monocyte-derived DCs, bone marrow-derived MSCs, and circulating lymphocytes obtained from healthy donors, as well as the laboratory-generated influenza virus hemagglutinin-derived peptide, aa 306–318 peptide-specific T cell line were used for this study. We demonstrate that MSCs mediate inhibition of DC function only upon cell–cell contact. Despite no http://www.jimmunol.org/ modification observed in cell phenotype or cytokine production, MSC-treated DCs were unable to form active immune synapses; they retained endocytic activity and podosome-like structures, typical of immature DCs. The transcriptional program induced by MSC– DC direct interaction supports at the molecular pathway level the phenotypical features observed, indicating the genes involved into contact-induced rearrangement of DC cytoskeleton. The Journal of Immunology, 2010, 185: 5102–5110.

one marrow mesenchymal stem cells (MSCs) are multi- cross-talk between MSCs and T cells may be responsible for T cell potent cells able to differentiate in vitro and in vivo into immunosuppression, and LPS stimulation may inhibit this sup- by guest on September 27, 2021 B tissues of mesenchymal and nonmesenchymal origin. pression by downregulating Jagged1 expression via TLR4 (4). MSCs have been extensively studied for their immunoregulatory However, even if MSC stimulation by LPS is followed by upreg- properties, and there is wide agreement that they inhibit in vitro ulation of TLR4 expression, it does not affect the ability of MSCs to T cell proliferation and dendritic cell (DC) immunogenicity and induce the differentiation of nonimmunogenic DCs (5). Conflicting can ameliorate in vivo autoimmune diseases, such as experimental results were reported for MSC/B cell interaction. Corcione et al. (6) allergic encephalomyelitis (1, 2). MSCs have been approved as showed MSC-mediated inhibition of the proliferation and differ- treatment for severe graft-versus-host disease (3). A Notch-Jagged1 entiation of human peripheral B cells into Ig-secreting cells; in contrast, a more recent study concluded that B cells can efficiently respond to the TLR9 agonist CpG 2006 in the absence of BCR triggering when cocultured with MSCs and that MSCs stimulate the *Department of Neurological Sciences, †Department of Pharmacology, and proliferation and differentiation of B into Ig-secreting cells isolated ‡Department of Anatomy, Histology and Forensic Medicine, University of Flor- ence; and xDepartment of Hematology, Careggi Hospital, Florence, Italy from patients affected by systemic lupus erythematosus (7). The extent to which the effects of MSCs on immune-system cells are Received for publication April 23, 2010. Accepted for publication August 20, 2010. mediated by soluble factors and/or cell–cell contact has yet to be This work was supported in part by the Italian Ministry of Education, Universities and Research (Grant 2007LTAJMA_003 to P.R.) and by the European Community fully understood. Different properties of MSCs may depend on the projects sixth frame program (FP6) Network of Excellence DC-THERA, EU LSHB- mechanism of stimulation, cell concentration, and type of coex- CT-2004-512074 and FP7 Integrative Project SYBARIS (Grant 242220), and MIUR isting immune cells. Progetti di Ricerca di Interesse Nazionale 519MIUR060 (to D.C.). Focusing our research on MSC/DC interactions during DC Address correspondence and reprint requests to Dr. Clara Ballerini, Department of Neurological Sciences, University of Florence, Viale Pieraccini, 6, 50139 Florence, activation, we show that upon direct physical interaction with Italy. E-mail address: clara.ballerini@unifi.it MSCs, DCs become unable to form active immune synapses with The online version of this article contains supplemental material. lymphocytes, despite their expression of a mature phenotype and Abbreviations used in this paper: BG, background; ctr, control; DC, dendritic cell; normal IL-12/IL-10–production profile. In contrast, MSC-treated DEG, differentially expressed gene; FAK, focal adhesion kinase; FluHA 316–318, DCs retain endocytic activity and podosome-like structures, typ- influenza virus hemagglutinin-derived peptide, aa 306–318; KEGG, Kyoto Encyclo- pedia of Genes and Genomes; MSC, mesenchymal stem cell; MYL9, myosin regu- ical of “immature” DCs. We also show that the behavior of MSC- latory L chain 9; MYLK, myosin L chain kinase; NIH, National Institutes of Health; treated DCs is accompanied by rearrangement of the cytoskeleton sBEF, signed binary enrichment factor; STRING, Search Tool for the Retrieval of and reprogramming of mRNA expression, as well as that cell–cell Interacting Genes/Proteins; tw, Transwell. contact, assessed by electron microscopy, is mandatory for MSC- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 mediated inhibition of DC immunogenicity. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001332 The Journal of Immunology 5103

Materials and Methods (1:200; Sigma-Aldrich); or monoclonal mouse anti-human Rac1 (2 mg/ml; Cells USBiological), followed by Alexa Fluor 488-conjugated rabbit anti-mouse IgG (1:200; Molecular Probes). DCs were generated from human monocytes of healthy donors, as pre- Confocal images were acquired using a Leica TCS SP5 microscope viously described (8). Briefly, anti-CD14+ monocytes were positively (Leica Microsystems, Mannheim, Germany) equipped with a He/Ne/Ar sorted by magnetic microbeads (Miltenyi Biotec, Bologna, Italy). Mono- laser source, using a Leica Plan Apo 363/1.40 NA oil-immersion objec- cytes were cultured for 6 d in medium supplemented with GM-CSF (1000 tive. A series of optical sections (1024 3 1024 pixels each; pixel size, 200 3 U/ml) and IL-4 (1000 U/ml; both from Labogen, Milano, Italy). Immature 200 nm) was taken at intervals of 0.35 mm. Confocal images were DCs were activated by 24 h of incubation with LPS (1 mg/ml; Sigma- deconvolved using ImageJ 3D deconvolution software (National Insti- Aldrich, St. Louis, MO) or 3 h of Ag loading (10 mg/ml, influenza virus tutes of Health [NIH], Bethesda, MD). Densitometric analysis was per- hemagglutinin-derived peptide, aa 306–318 [FluHA 306–318], kindly formed with ImageJ software (NIH), measuring the average fluore- provided by Anna Maria Papini, PeptLab, Department of Organic Chem- scence intensity on regions of interest in single focal-plane images. istry, University of Florence). CD4+ T cells were negatively selected from PBMCs using the T cell isolation kit II from Miltenyi Biotec. The FluHA T cell-proliferation tests 306–318–specific T cell line was generated in our laboratory through semicloning methodology (9). Ag specificity was tested periodically. MLR was performed in 96-well U-bottom plates (Nunc, Roskilde, Danemark). 3 5 + MSCs were obtained as previously reported (5). Briefly, whole bone A total of 1 10 CD4 T cells were cultured for 5 d in RPMI 1640 with 10% 3 4 marrow was collected from the iliac crest in tubes containing acid citrate FCS, together with 1 10 allogeneic DCs/well activated with LPS, with or dextrose and centrifuged for 10 min at 700 3 g; the interface between without MSCs. Experiments were conducted in quadruplicate. At day 5, the proliferative response was measured by [3H]thymidine (1 mCi/ml; Amersham plasma and red cell pellet (buffy coat) was recovered and diluted 1:10 in 3 HBSS without calcium and magnesium (Euroclone, Milano, Italy) to be Bioscience, Piscataway, NJ) incorporation test. [ H]thymidine was added for 2 3 5 2 the last 8 h of culture. Plates were harvested (Tomtec Mach III, Wallac, Turku, counted. Cells were then plated in 75-cm flasks (1.6 10 cells/cm )in 3 DMEM with low glucose (Life Technologies, Invitrogen, Milano, Italy); Finland) on glass fiber filters (PerkinElmer, Wellesley, MA), and [ H]thy- Downloaded from 10% FBS (HyClone, Milano, Italy) was added, and the cells were in- midine uptake was measured by liquid scintillation in a Microbeta 1450 3 5 cubated at 37˚C in humidified atmosphere containing 95% air and 5% Trimux counter (Wallac). The FluHA 306–318–specific T cell line (1 10 cells/well) was tested for the proliferative response to autologous DCs (1 3 CO2. At confluence, the adherent cells were harvested with 0.05% trypsin 3 and 0.02% EDTA (Eurobio, Toulouse, France) and resuspended in com- 10 cells/well) loaded with peptide Ag for 3 h, with or without the presence of plete medium (primary culture). Cells were plated again at 104 cells/cm2 in MSCs; where indicated, MSCs were pretreated with anti–VCAM-1 or anti–N- 100-mm dishes, and expansion of the cells was obtained with successive cadherin blocking Abs, as previously described. After 48 h, the proliferative cycles of trypsinization and reseeding. Cells were used at different pas- response was evaluated as above. Transwell experiments were performed in m http://www.jimmunol.org/ sages. Two DC/MSC ratios, 10:1 and 1000:1, were used in all experiments. Transwell permeable supports (0.4- m polyester membrane; Costar, Cam- Before testing their stimulatory function, DCs cocultured with MSCs were bridge, MA). negatively selected from coculture by labeling with PE-conjugated CD105 T cells were recovered after 5 d of culture in an MLR performed with Ab, followed by incubation with anti-PE magnetic microbeads (Miltenyi DCs activated with or without the presence of MSCs, as described above. 3 5 Biotec). After 6 h of resting at 4˚C, T cells (1 10 cells/well) were stimulated with All human participants gave written informed consent, and the study IL-2 (50 U/ml) and tested for subsequent proliferative responses. was authorized by the local ethical committee (protocol number 2/99 18.264-04). Electron microscopy and immunoelectron microscopy DCs were activated with LPS for 24 h in the presence of MSCs, and the Immunocytochemistry cocultures were fixed, while attached to flasks, with 2% (w/v) formaldehyde by guest on September 27, 2021 To analyze immune synapse formation between Ag-specific T cells and and 2.5% glutaraldehyde in 0.1 mol L-1 cacodylate buffer (pH 7.4) for Ag-loaded DCs, DCs were loaded with FluHA 306–318 peptide for 3 h, 30 min for conventional microscopy and for 10 min for immunoelectron with or without the presence of MSCs. In one experimental condition, microscopy, at room temperature. Then they were removed with a scraper MSCs were pretreated with purified azide-free mouse monoclonal Abs and pelleted. For immunoelectron microscopy, endogenous peroxidases against VCAM-1 (clone P3C4; Santa Cruz Biotechnology, Santa Cruz, were blocked with 0.3% H2O2 in PBS for 30 min. CD43 and CD54 CA; used at 30 mg/ml following the manufacturer’s instructions) and were tagged with mouse monoclonal Abs (BD Pharmingen), followed by N-cadherin (clone GC-4; Sigma-Aldrich; used at 80 mg/ml following the peroxidase-labeled polyclonal goat anti-mouse Abs (1:100, Sigma- manufacturer’s instructions); after overnight incubation at 37˚C with Aldrich), and visualized with diaminobenzidine. In all cases, cells were blocking Abs, MSCs were washed twice in fresh medium and cocultured osmicated and embedded in epoxy resin. Sections were observed using with DCs. In agreement with results reported by Ren et al. (10), this ex- a Jeol 1010 electron microscope (Tokyo, Japan) at 80 kV. Immunolabe- perimental condition functionally reduces the MSC/DC cell–cell adhesion led cells were observed unstained; otherwise, the sections were stained by 30%. This is the mean value from two experiments in which adhesion with uranyl acetate and lead citrate (Polysciences, Warrington, PA). was evaluated as the number of DCs in contact with MSCs in six micro- Densitometry. Labeled cell membranes in experiments with LPS were scope fields randomly selected by a 203 objective (field size was ∼0.9 3 subjected to densitometry, by outlining thin (2–5-mm wide) strips on the 0.9 mm). After Ag loading and negative separation from MSCs, DCs were digitalized photomicrographs and analyzing them with the macro for gel reseeded on coverslips (3 3 104 cells) and left to adhere for 30 min at 37˚C, analysis by Image 1.63 software (NIH). The gray level of cytoplasm was then an equal number of T cells was added. After 45 min at 37˚C, T cell/DC subtracted from that of all peaks, and the total area of each peak corre- conjugates were washed with PBS, fixed with 4% phosphate buffered sponding to a plasma membrane was assumed as the intensity of labeling paraformaldehyde for 10 min, permeabilized with 0.05% saponin (ICN of that membrane, in arbitrary units. Biomedicals, Zurich, Switzerland) and 2% BSA (Sigma-Aldrich) in PBS, Statistics for densitometry. Because the intensity of labeling was widely and labeled with monoclonal mouse anti-human CD43 Ab (10 mg/ml; BD distributed and not all of the membranes appeared labeled, a two-step Pharmingen, San Diego, CA), followed by rabbit anti-mouse IgG Alexa statistical analysis was applied. The data for each experimental condition Fluor 546 (1:200; Molecular Probes, Eugene, OR) or were diluted in PBS, and cell type (i.e., MSCs versus DCs, independent of the cell side) were 2%BSA,and0.05%saponinandthenincubatedfor2and1hatroom first analyzed by the x2 test, to verify whether the number of la- temperature, respectively. beled cells depended on the cell type. Upon visual inspection of data To analyze actin/Rac1/PAK1 distribution, DCs preincubated with or showing skewness, the data relative to the labeled membranes were without the presence of MSCs were seeded on glass coverslips (5 3 104 transformed into their logarithms and subjected to ANOVA among the cells) after 24 h of LPS stimulation for actin labeling and after 5 min of different cell types and the different surfaces of DCs (i.e., whether or LPS stimulation for Rac1 and PAK1 labeling. Cells were fixed and per- not they were facing another cell) for each tagged membrane molecule. meabilized, as described above, for immune synapse analysis. Actin was When this analysis gave significant results, the Student t test for unpaired labeled with FITC-conjugated phalloidin (0.1 mg/ml; Sigma-Aldrich) for values was used to check which values diverged from one other. All tests 30 min at room temperature. Primary and secondary Abs were diluted in applied were two tailed, and p , 0.01 was considered significant (the PBS with 2% BSA and 0.05% saponin and incubated for 2 and 1 h, re- relatively low value of p needed for significance was selected because of spectively. The following Abs were used: monoclonal mouse anti-human the large number of comparisons). Because of the logarithmic trans- CD11c, PE conjugated (1:50; BD Pharmingen); monoclonal rabbit anti- formation of the intensities of labeling, the corresponding results are human PAK1 (2 mg/ml; USBiological, Milano, Italy), followed by tetra- given as median 6 SE of the median, and the SE is asymmetrically methylrhodamine isothiocyanate-conjugated monoclonal mouse anti-rabbit distributed around the median (11). 5104 DC/MSC CONTACT: A NEW PATHWAY OF IMMUNE REGULATION

DC surface markers signatures and signed binary enrichment factors (sBEFs). sBEFs were clustered, using bootstrapping with support trees (17). This computation DC surface markers were evaluated by flow cytometry with a four-color was carried out with the TIGR Multiexperiment Viewer (TmeV, Dana- EpicsXL cytometer (Beckman Coulter, Fullerton, CA), equipped with Farber Cancer Institute, Boston, MA) 4.4. Expo 32 software. The Abs used (Beckman Coulter) were directed against the following Ags (the tags are given in parentheses): CD80 (FITC), CD86 (PE), HLA-DR (ECD), and CD83 (PC5). Cell vitality was tested with Results propidium iodide (Molecular Probes). The cells were labeled in PBS with MSCs inhibit the ability of DCs to perform active immune 1% FCS for 25 min at 4˚C, washed twice, and analyzed immediately. synapses after short-term coculture Cytokine determination Standard MLR was used to assess the ability of DCs cocultured DCs were activated with LPS (1 mg/ml) for 24 h, with or without MSCs; with MSCs during LPS activation to stimulate CD4+ T cells (Fig. the supernatants were collected and stored at 280˚C for cytokine de- 1A). Upon coculture with MSCs, DCs were no longer able to in- termination by ELISA (Quantikine kit, R&D Systems, Minneapolis, MN). duce T cell-proliferative responses. To address whether this result Dextran uptake depended on soluble factors or cell–cell contact, we cocultured Endocytosis by resting or LPS-activated DCs was examined as described by MSCs and DCs in contact with each other or in a Transwell system Sallusto et al. (12). Briefly, 1 3 105 cells were resuspended in RPMI 1640 and stimulated them with LPS for 24 h. The three cell groups with 10% FCS, equilibrated at 37˚C or 0˚C for 10 min, and pulsed with (control without MSCs, Transwell coculture, and contact coculture) FITC-conjugated dextran (40,000 Da; Molecular Probes), 1 mg/ml for were tested by a classical MLR assay (Fig. 1B): DCs activated in 45 min at 37˚ or 0˚C. Cells were washed four times with cold PBS and an- contact with MSCs were virtually unable to stimulate the lympho- alyzed by flow cytometry; propidium iodide was used to exclude dead cells. The labeling of cells pulsed at 0˚C was subtracted from that of cells pulsed cytes, whereas the stimulation provided by DCs activated in a Downloaded from at 37˚C to obtain a measure of the endocytosed dextran. Transwell was similar to that of controls. After 5 d of MLR, as de- scribed in Fig. 1B, the T cells were recovered and tested for pro- Transcriptional analysis liferative response to IL-2 stimulation. The T cells previously 6 Atotalof13 10 DCs were cocultured with MSCs at a ratio of 10:1 in the allostimulated by DCs activated in contact with MSCs did not presence of LPS; alternatively, DCs or MSCs were cultivated with LPS. After proliferate, even upon IL-2 challenge (Fig. 1C). The ability to re- 4 h, cells were collected. RNA preparation was performed with TRIzol reagent. Labeling, hybridization on a HT12 WholeGenome BeadArray (Il- verse MSC inhibition by blocking adhesion molecules VCAM-1 http://www.jimmunol.org/ lumina, San Diego, CA), and scanning were performed according to the and N-cadherin was tested during Ag-specific T cell line stimula- Illumina reference protocols. The analysis was performed on three donors. tion. We compared T cell proliferation after stimulation with au- Array preprocessing tologous DCs loaded with Ag in the presence or absence of MSCs pretreated with anti–VCAM-1 and anti–N-cadherin Abs (Fig. 1D) Bead-summary data saved from Illumina BeadStudio were preprocessed in (see Materials and Methods). Our data show that the tolerogenic several steps. The background signal was assessed and corrected using the intensity signal from the control probes present on the array and then effect of MSCs on DCs was significantly reversed in the presence of quantile normalization was performed. In addition to background correc- Abs blocking adhesion molecules (one-sample t test; p = 0.003). tion, Illumina probe identifiers were converted to nucleotide universal The ability of mature DCs to engage in active immune synapses identifiers (13) specific for the nucleotide sequence of each probe. The with T cells is instrumental to inducing T cell activation. To address by guest on September 27, 2021 computation was performed using the lumi package (14) written in the R programming language. whether DCs loaded with Ag in the presence of MSCs are able Data were submitted to Array Express under accession number E- to establish immune synapses during lymphocyte stimulation, we MTAB-286 (http://www.ebi.ac.uk/microarray-as/ae/). analyzed CD43 distribution at the contact surface between DCs and Differential expression, annotation, and visualization a FluHA 306–318–specific T cell line by confocal microscopy. When FluHA 306–318 cells were challenged with peptide-loaded Differential expression analysis was carried out using the Rank Product DCs, we observed the formation of active immune synapses, algorithm (15), taking into account the differences among donors. The p values estimating differential expression were corrected for multiple where CD43 underwent relocalization away from contact areas in testing, and genes with a corrected p value #0.05 were selected. Dif- almost all of the cell conjugates analyzed (Fig. 2A, upper left ferentially expressed gene (DEG) lists from the various data sets were panels). On the contrary, DCs cocultured with MSCs contacted uploaded in the Database for Annotation, Visualization and Integrated lymphocytes on areas resembling immune synapses but did not Discovery (http://david.abcc.ncifcrf.gov) to perform functional anno- undergo relocalization of CD43 (Fig 2A, upper right panels), as tation. The upregulated and downregulated gene lists were annotated separately. Gene Ontology enrichment over Biological Process was confirmed by densitometry analysis (Fig. 2A, lower panels). To performed using the Biological Network Gene Ontology plug-in for the evaluate the direct mechanistic effect of adhesion in this phe- Cytoscape Desktop. To perform sample and gene clustering, a matrix nomenon, we performed DC Ag loading in the presence of with the absolute expression of the differentially expressed genes was MSCs previously treated with blocking Abs against VCAM-1 and extracted from the normalized data and then clustered using the Pearson correlation, bootstrapping genes, and samples over 1000 iterations. N-cadherin. We counted the number of active immune synapses Visualization of the genes on a subset of Kyoto Encyclopedia of Genes per microscope field, using an 340 objective, in six randomly and Genomes (KEGG) pathways was performed using the KEGG web selected fields (Fig. 2B); field size was ∼387 3 387 mM. The service, with the aid of in-house programs. mean number of active synapses per field (6 SD) was 5.6 6 1.2 Identification of predicted relationships in the control sample, which decreased to 3.2 6 0.6 (p = 0.049) when peptide loading was done in the presence of MSCs. When The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) Web site (http://string.embl.de) was used to infer knowledge about the known the MSCs were preincubated with anti–VCAM-1 or anti–N- interactions of the DEGs annotated in KEGG pathways. For each pathway, cadherin blocking Abs, the mean number of active synapses was lists of gene symbols representing DEGs in that pathway were uploaded into 6 6 0.9 and 4.2 6 0.6, respectively. Pretreatment with anti– STRING, and the minimum confidence score was set to medium (0.4). VCAM-1 Abs significantly reversed (one-sample t test, p = 0.032) Pathway analysis the inhibitory effect of MSCs on DC Ag-presentation capability. Pathway analysis was performed using the procedure of Beltrame et al. (16). MSCs and DCs: two ways to be in contact Briefly, the Fisher Exact Test was run over paired case-control ratios (cocultures versus single cells) calculated from the normalized data, using Because DCs and MSCs appeared to adhere to each other during the public pathway set. Then, p values were transformed into pathway coculture (Fig. 3A), electron microscopy and immunoelectron The Journal of Immunology 5105 Downloaded from http://www.jimmunol.org/

FIGURE 1. MSCs inhibit DC stimulatory function through cell–cell contact. A, MLR: proliferative response of CD4+ T cells stimulated by allogeneic DCs activated with LPS in the presence of MSCs (DC/MSC ratio, 10:1) or without MSCs (ctr). B, MLR as in A performed in a Transwell system (tw): proliferative response of T cells stimulated by DCs activated in contact with MSCs (gray bars), by DCs cocultured with MSCs in a tw (white bars), or by by guest on September 27, 2021 control DCs (black bars). C, T cell recovery after MLR: T cells stimulated as in B at day 5 of MLR were selected, washed, and stimulated with IL-2. Data in A–C are representative of one of three independent experiments; the proliferative response is expressed as mean cpm of four wells 6 SD. D, T cell line stimulation by DCs loaded with Flu in coculture with MSCs or with MSCs previously treated with anti–VCAM-1 or anti–N-cadherin blocking Abs or without MSCs (ctr). Data are expressed as mean values of three independent experiments. BG, background. microscopy were used to analyze the contacts between these DCs do not undergo RAC-1 and actin redistribution upon two cell types. Using electron microscopy, contact zones be- MSC/DC short-term interaction tween MSCs and DCs were mainly suggestive of adherens junc- Actin distribution is regulated by small GTPases of the Rho family, tions (Fig. 3B). Rarely, we found contact zones suggestive of such as Rac1, which has PAK1 as an immediate target and is gap junctions (Fig. 3C). The expression of CD54, on average, responsible for polarization-related morphological changes. Rac1 , was significantly (p 0.01) less intense on the surfaces of DCs is upregulated by LPS exposure, and activation of Rac1 is asso- in contact with MSCs than on the rest of the cell surface, ciated with its rapid recruitment into lipid rafts and podosome whereas CD43 was equally distributed on the surfaces of DCs, disassembly. Because actin redistribution is involved in endocy- suggesting that upon contact between MSCs and DCs, the latter tosis, we addressed whether coculture with MSCs during DC ac- cells undergo only partial redistribution of the CD54 molecule tivation by LPS influences the behavior of this protein. As shown alone (Fig. 4). by confocal microscopy, DCs activated by LPS without MSCs MSC/DC short-term interaction interferes with DC endocytosis completely lost their podosomes, which are typical of immature but does not influence DC phenotype and cytokine production DCs, and showed uniform distribution of actin along the cell surface (Fig. 6A, a–d). On the contrary, MSC-treated DCs retained We investigated what influence, if any, coculture with MSCs during structures that were suggestive of podosomes, as shown by colo- DC activation had on DC phenotype, cytokine production, and calization of actin and CD11c (Fig. 6A, e–h). endocytosis. The cell phenotype, expressed as the percentage DCs activated without MSCs expressed and concentrated Rac1 of cells positive for CD80, CD83, CD86, and HLA DR, did not together with PAK1 along the cell surface (Fig. 6B, i–l), whereas change when activation took place in the presence of MSCs (Fig. Rac1 was minimally expressed, and PAK1 remained distributed in 5A). Endocytosis, as evaluated by dextran-FITC uptake, decreased the cytoplasm of MSC-treated cells (Fig. 6B, m–p). upon activation in the absence of MSCs, whereas this decrease was not observed upon DC activation in the presence of MSCs (Fig. 5B). The production of IL-10 and IL-12, two key cytokines Transcriptional sensing of DCs upon contact with MSCs produced by DCs, did not vary significantly upon activation in the To gain insight into the molecular mechanisms subtending DC presence of MSCs (Fig. 5C,5D). reprogramming by MSCs, we investigated the transcriptional 5106 DC/MSC CONTACT: A NEW PATHWAY OF IMMUNE REGULATION Downloaded from http://www.jimmunol.org/ FIGURE 2. Immune synapses between DCs and T cells analyzed for CD43 distribution using confocal microscopy. A, Left panels, Control (ctr): active immune synapse with CD43 distributed far from the synaptic area; the graph shows anti-CD43 OD (arbitrary units) against conjugate length; maximum OD was measured (arrow). Right panels, Upon activation of DCs in the presence of MSCs, CD43 remains mainly distributed at the cell–cell contact zone. Images are representative of 40 conjugates analyzed in two independent experiments (original magnification 360). B, Mean number of active synapses/field counted in six different fields randomly selected at 340 magnification. Black bar, control; white bar, DCs activated in the presence of MSCs; striped bar, DCs activated in the presence of MSCs preincubated with anti–VCAM-1 Abs; stippled bar, DCs activated in the presence of MSCs preincubated with anti– N-cadherin Abs. Data are representative of one of two independent experiments. by guest on September 27, 2021 response of DCs to cell contact with MSCs in the presence of LPS the regulation of pathways and cellular networks in our samples, (see Materials and Methods). We performed microarray analysis we performed a pathway analysis (16) and clustered the results as by comparing DCs cocultured with MSCs with DCs or MSCs sBEFs (Supplemental Fig. 1). The transcriptional changes induced cultured alone, in triplicate. In this way, we obtained a statistically by the contact of MSCs with DCs were dissected out by focusing robust list of DEGs; in particular, 422 genes resulted differentially on the comparison between the coculture condition and the DCs expressed in the cocultures with respect to DCs alone and 403 cultured alone by means of the Pearson correlation. As previously with respect to MSCs alone (Supplemental Table I). To investigate described, we identified clusters of genes whose expression was differentially modulated in the presence or absence of MSCs. Among them, several genes were mapped on KEGG pathways, which resulted significantly affected according to a false discovery rate-corrected Fisher exact test (p , 0.05), in particular the regu- lation of actin cytoskeleton, adherens junctions, gap junctions, and tight junctions (Fig. 7A, Supplemental Fig. 2). Among the members of the actin cytoskeleton-regulation pathway, we observed an increased expression of the genes coding for integrins ITGA11 (4.21-fold change) and ITGB5 (6.04-fold change), as well as fibro- nectin (FN1; 3.09-fold change). In addition, we observed an up- regulation of genes coding for focal adhesion kinase (FAK) (PTK2; 2.38-fold change), the noncatalytic region of tyrosine kinase adaptor protein-associated protein Nap125 (NCKAP1; 2.95-fold change), myosin L chain kinase (MYLK) (MLCK; 2.60-fold change), and myosin regulatory L chain 9 (MYL9) (MLC; 2.62-fold change). The upregulation of MYLK and MYL9 is in agreement with an upreg- ulation of myosin II (MYH10; 3.61-fold change), part of the same chain of reactions. Among genes mapped on adherens, gap, and FIGURE 3. DC/MSC contacts. A, Phase-contrast microscopy. B–D, tight-junction pathways, we found increased expression of nectin 3 transmission electron microscopy. B, Two DCs (D) in contact with an MSC (PVRL3; 4.09-fold change), connexin 43 (Cx43; 23.36-fold change), (M). C, Close contact suggestive of a gap junction (circle). D, Adherens and coractin (CTTN; 2.99-fold change), respectively. junction (square) with an adhesion plaque and microfilament bundles on the In contrast, the LPS pathway was not influenced by the presence MSC side and electron-dense material in the 35-nm intermembrane space. of MSCs, as evidenced by the lack of over- or downregulation of The Journal of Immunology 5107 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 FIGURE 4. Contact (arrow) between MSCs (M) and DCs (D) leads to modifications in CD54 (arrowhead), but not CD43, membrane expression and distribution, as shown by pre-embedding immunoelectron microscopy (left panels). Densitometry evaluation of labeling (median 6 SE) (right panels). p not significant for CD43; pp , 0.01 for CD54. involved genes in the coculture condition, with respect to DCs conditions (18–23). It was suggested that a shift from soluble alone. factors to cell–cell contact mechanisms is associated with MSC Known interactions between our DEGs and other genes strongly dilution (22), which was high (MSC/DC cell ratio 1:10 and suggest that nectins and nectin-like molecules could interact with 1:1000) in our experimental setting. Electron microscopy images FAK (PTK2), which, in turn, cooperates with integrins in actin showed that MSCs and DCs came in intimate contact with each cytoskeleton organization (Fig. 7B). other stabilized by adherens junctions, accompanied by the uneven distribution of CD54, but not of CD43, on DCs, indicating that Discussion these contacts cannot be assimilated to immune synapses (24). The extent to which effects of MSCs on immune system cells are These findings, together with the unaltered costimulatory mole- mediated by soluble factors and/or cell–cell contact is still being cule expression and cytokine production by DCs, suggest that the debated. Elucidating this fundamental issue is crucial to under- inability of MSC-treated DCs to engage in active synapses with standing how different properties of the DC interaction with MSCs lymphocytes is an active process rather than the expression of an depends on cell concentration and localization. exhausted or damaged cell phenotype (25). Our results are extremely innovative in this field of research. In Data from other studies showed that, in humans and mice, TNF- the present work, we show how fully differentiated DCs activated in a– or LPS-stimulated maturation of DCs in the presence of MSCs contact with MSCs induce stable not responsiveness of viable CD4+ modifies the expression of costimulatory molecules needed for the T cells. Further, we demonstrate that this depends on the inability of activation of lymphocytes (4, 23, 25–27). Our data showed that DCs to establish active immune synapses with lymphocytes. This MSC-treated DCs maintain endocytic activity typical of the im- inability is associated with other features of immaturity retained by mature phenotype, indicating that LPS activates membrane mol- DCs: altered Rac1 redistribution and alteration in the gene networks ecule and cytokine expression, whereas MSC contact inhibits full responsible for actin cytoskeleton organization. cell maturation. The process by which DCs move from the im- We studied the immunomodulatory effects of MSCs cocultured mature phenotype suitable for Ag acquisition to the mature one with DCs during LPS activation or Ag loading, and we showed by suitable for Ag presentation is a broader change that involves Transwell experiments that the tolerogenic effect of MSCs on more than mere upregulation of MHC class II and CD86 mem- DCs is dependent on cell–cell contact interactions, not on soluble brane molecules; it also includes reorganization of the cytoskel- factors (i.e., IL-6 and PGE2), as reported in other experimental eton (28). A recent model proposed DC activation as the integra- 5108 DC/MSC CONTACT: A NEW PATHWAY OF IMMUNE REGULATION Downloaded from http://www.jimmunol.org/

FIGURE 5. MSC/DC short-term interaction interferes with DC endocytosis but does not influence DC immunophenotype or cytokine production. A, Immunophenotype. B, Flow-cytometry measure of endocytosis: labeling without LPS activation (upper panels) and labeling after LPS activation (lower panels). Filled curve, negative control. ELISA determination of IL-10 (C) and IL-12 (p40+p70) (D); each circle represents a sample. tion of many stimuli not in a linear system (29). In our experiments, immature, not immunogenic, DCs (30). On the contrary, cells MSC-treated DCs showed colocalization of actin with CD11c, activated without MSCs lose these structures and have actin dis- indicating the presence of podosome-like structures typical of tributed all around the cell, as expected for mature DCs (31). by guest on September 27, 2021

FIGURE 6. Coculture with MSCs during activation affects the organization of DC cytoskeleton. Confocal laser-scanning microscopy of DCs activated with LPS but without MSCs (controls; a–d, i–l) or in the presence of MSCs (e–h, m–p). A, Green, F-actin; red, CD11c. B, Green, Rac1; red, PAK1. Colocalization as determined by xz view is depicted in d, h, l, and p. Original mag- nification 360. The Journal of Immunology 5109 Downloaded from http://www.jimmunol.org/

FIGURE 7. DEGs related to DC–MSC cell contact and prediction relationship. A, DEGs involved in actin cytoskeleton, adherens junction, gap junctions, by guest on September 27, 2021 and tight junctions. B, Prediction relationships obtained using the STRING Web site.

Actin distribution is regulated by small Rho GTPases (32), and it ulation of the gene for FAK which, in addition to mediating the was reported that Rac1 is activated shortly after LPS stimulation response to adhesion to the extracellular matrix (35), is involved in (33). We showed that 5 min of LPS stimulation, in the absence of the formation of intercellular adherens junctions and in the ac- MSCs, led to the activation of Rac1 and PAK1 and that this tivation of adhesion-dependent signal pathways, including actin process was inhibited in MSC-treated DCs. These findings suggest network rearrangement (36, 37). Known interactions between the that the induction of nonimmunogenic DCs depends on inter- DEGs found in this study and other genes strongly suggest that ference with rearrangement of the cytoskeleton during or upon nectins and nectin-like molecules could interact with FAK (PTK2) activation. Together with the description of a new DC-regu- to drive actin cytoskeleton organization. In this scenario, DCs and lation mechanism through adhesion, we attempted to further un- MSCs seem to use the nectin-based cell–cell adhesion to sub- derstand the possible molecules involved. For this purpose, we sequently reorganize actin cytoskeleton. performed a study of mRNA transcription, focusing on expression The observation that the expression of the genetic networks pathways specific for adhesion and activation. In each condition, induced by the LPS pathway is not affected by the presence of we exposed the cells to LPS to understand the role of MSCs in MSCs indicates that the MSC messages are primarily mediated by controlling DC immune response and to consider the effect of LPS molecules that are part of the cytoskeleton and cell–cell junctions, on the transcriptional program. The signaling pathways described and, therefore, are influenced solely by contact rather than by in the Results are consistent with our imaging and functional data other soluble signals. Strikingly, this gene-expression result is fully and support the idea that MSC immunomodulation acts through in agreement with the other cell-based assays, indicating the vali- several molecules involved in adhesion, as confirmed by our dity of our observation. adhesion-blocking experiments. Indeed, the importance of adhe- It is known that actin reorganization requires Rac signaling, sion and adhesion molecules in the immunomodulatory action a pathway that is thought to be mediated by the protein Scar/WAVE of MSCs on T cells was demonstrated recently by Ren et al. (Wiskott-Aldrich syndrome protein-family verprolin homologous (10). They showed that ICAM-1 and VCAM-1 are required for protein) (38, 39). The absence of Rac1 expression, in association lymphocyte–MSC adhesion and play an important role in MSC- with the upregulation of Nap125 (NCKAP1 gene), whose function mediated immunosuppression. We found upregulation of connexin is to inhibit the WAVE1/HSPC300 complex, could explain the 43 (Cx43) gene, a major gap junction protein, and the adhesion lack of proper cytoskeleton reassembling that we observed. More- receptor nectin 3, which is primarily involved in the regulation over, the absence of PAK expression took into account the ex- of cell–cell adhesion and contact-dependent inhibition of cell pression of MYLK and MYLK-associated genes (MLC) on myosin movement and proliferation (34). Moreover, we found upreg- expression. 5110 DC/MSC CONTACT: A NEW PATHWAY OF IMMUNE REGULATION

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Supplemental Fig. 1. Clustering of signed Binary Enrichment Factor (sBEFs) obtained from the

FET analysis on co-culture samples with respect to DCs or MSCs. Coloured spots indicate significant (p, 0.05) up- (red) or down- (green) regulation. The colours of the dendrogram indicate the percentages of the tree support (significance), from 50% (pink) to 100% (black).

Supplemental Fig. 2. Visualization of differentially expressed genes (DEGs) related to DC-MSC cell contact on KEGG pathway. DEGs were mapped in KEGG actin cytoskeleton organization, adherens junction, gap junctions, tight junctions. The differentially regulated genes are indicated in red.

DCs + MSCs LPS vs DCs LPS

Gene Symbol Gene Name Fold Change ABI3BP ABI family, member 3 (NESH) binding protein 17.3611111111111 ACOX2 acyl-Coenzyme A oxidase 2, branched chain 2.68744961031981 ACTA2 actin, alpha 2, smooth muscle, aorta 3.95882818685669 ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 6.16142945163278 ADAMTS5 ADAM metallopeptidase with thrombospondin type 1 motif, 5 2.28414801279123 AHNAK2 AHNAK nucleoprotein 2 2.84010224368077 ALDH18A1 aldehyde dehydrogenase 18 family, member A1 2.34631628343501 ALDH1A3 aldehyde dehydrogenase 1 family, member A3 14.7275405007364 ALDH7A1 aldehyde dehydrogenase 7 family, member A1 2.18818380743982 ALPK2 alpha-kinase 2 2.55885363357216 ANGPTL2 angiopoietin-like 2 2.30096640589047 ANKRD1 ankyrin repeat domain 1 (cardiac muscle) 3.96353547364249 ANKRD29 ankyrin repeat domain 29 2.44558571777941 ANTXR1 anthrax toxin receptor 1 7.34753857457752 APCDD1L adenomatosis polyposis coli down-regulated 1-like 8.23045267489712 ARMCX2 armadillo repeat containing, X-linked 2 7.5642965204236 ARSJ arylsulfatase family, member J 2.24769611148573 ASAM adipocyte-specific adhesion molecule 7.55857898715042 ASS1 argininosuccinate synthetase 1 2.74122807017544 ATP8B2 ATPase, class I, type 8B, member 2 3.35457900033546 ATP9A ATPase, class II, type 9A 4.21762969211303 AXL AXL receptor tyrosine kinase 3.09310238168883 BACE2 beta-site APP-cleaving enzyme 2 2.66169816342827 BEX1 brain expressed, X-linked 1 2.56278831368529 BGN biglycan 41.1522633744856 BMP2 bone morphogenetic protein 2 3.52112676056338 C10orf116 chromosome 10 open reading frame 116 6.62251655629139 C11orf70 chromosome 11 open reading frame 70 2.16825672159584 C11orf70 chromosome 11 open reading frame 70 3.01477238468496 C12orf23 chromosome 12 open reading frame 23 2.65957446808511 C12orf24 chromosome 12 open reading frame 24 2.33863423760524 C13orf33 chromosome 13 open reading frame 33 4.03877221324717 C14orf37 chromosome 14 open reading frame 37 2.24114746750336 C14orf45 chromosome 14 open reading frame 45 2.12269157291446 C1QTNF5 C1q and tumor necrosis factor related protein 5 3.15855969677827 C1S complement component 1, s subcomponent 2.53613999492772 C5orf13 chromosome 5 open reading frame 13 4.02414486921529 C5orf23 chromosome 5 open reading frame 23 2.71886895051659 C5orf46 chromosome 5 open reading frame 46 3.10366232153942 C7orf10 chromosome 7 open reading frame 10 3.24464633354964 CA12 carbonic anhydrase XII 4.05350628293474 CALB2 calbindin 2 4.62107208872458 CALD1 caldesmon 1 4.40917107583774 CAMK2N1 calcium/calmodulin-dependent protein kinase II inhibitor 1 6.53167864141084 CAP2 CAP, adenylate cyclase-associated protein, 2 (yeast) 3.66434591425431 CAV1 caveolin 1, caveolae protein, 22kDa 3.13087038196619 CAV2 caveolin 2 4.41111601235112 CCDC34 coiled-coil domain containing 34 2.28675966155957 CCL2 chemokine (C-C motif) ligand 2 2.90107339715695 CCL7 chemokine (C-C motif) ligand 7 2.32558139534884 CCND1 cyclin D1 11.7096018735363 CD248 CD248 molecule, endosialin 6.23052959501558 CDH11 cadherin 11, type 2, OB-cadherin (osteoblast) 10.4058272632674 CDH13 cadherin 13, H-cadherin (heart) 2.62743037309511 CDH6 cadherin 6, type 2, K-cadherin (fetal kidney) 2.82565696524442 CHPF chondroitin polymerizing factor 2.79955207166853 CKAP4 cytoskeleton-associated protein 4 2.41545893719807 CLIP3 CAP-GLY domain containing linker protein 3 5.87889476778366 CMBL carboxymethylenebutenolidase homolog (Pseudomonas) 3.08166409861325 CNN3 calponin 3, acidic 8.01924619085806 CNPY4 canopy 4 homolog (zebrafish) 2.33644859813084 COBLL1 COBL-like 1 2.30574129582661 COL11A1 collagen, type XI, alpha 1 2.89519397799653 COL12A1 collagen, type XII, alpha 1 2.63019463440295 COL16A1 collagen, type XVI, alpha 1 2.62536098713573 COL1A1 collagen, type I, alpha 1 11.7233294255569 COL1A2 collagen, type I, alpha 2 42.7350427350427 COL3A1 collagen, type III, alpha 1 12.8865979381443 COL4A1 collagen, type IV, alpha 1 12.0192307692308 COL5A1 collagen, type V, alpha 1 18.348623853211 COL5A2 collagen, type V, alpha 2 22.5225225225225 COL6A1 collagen, type VI, alpha 1 3.45901072293324 COL6A2 collagen, type VI, alpha 2 4.66200466200466 COL6A3 collagen, type VI, alpha 3 33.7837837837838 COL7A1 collagen, type VII, alpha 1 2.97973778307509 COL8A1 collagen, type VIII, alpha 1 7.097232079489 COMP cartilage oligomeric matrix protein 3.72856077554064 COX7A1 cytochrome c oxidase subunit VIIa polypeptide 1 (muscle) 5.249343832021 CPA4 carboxypeptidase A4 2.50438266967193 CRIP2 cysteine-rich protein 2 4.96770988574267 CRISPLD2 cysteine-rich secretory protein LCCL domain containing 2 2.10216523018709 CRYAB crystallin, alpha B 18.2149362477231 CTGF connective tissue growth factor 14.9031296572280 CTHRC1 collagen triple helix repeat containing 1 7.5187969924812 CTSK cathepsin K 10.5596620908131 CTTN cortactin 2.99490865528601 CXCL11 chemokine (C-X-C motif) ligand 11 3.02480338777979 CXCL12 chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 3.30687830687831 CXCL6 chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) 3.45661942620118 CXCR7 chemokine (C-X-C motif) receptor 7 2.24164985429276 CYGB cytoglobin 7.10732054015636 CYR61 cysteine-rich, angiogenic inducer, 61 2.54971953085161 DBC1 deleted in bladder cancer 1 3.5486160397445 DBN1 drebrin 1 4.18235048097030 DCBLD2 discoidin, CUB and LCCL domain containing 2 12.9701686121920 DCN decorin 32.7868852459016 DDAH1 dimethylarginine dimethylaminohydrolase 1 5.77700751010976 DDR2 discoidin domain receptor tyrosine kinase 2 4.04367165386171 DKK3 dickkopf homolog 3 (Xenopus laevis) 10.9649122807018 DLX5 distal-less homeobox 5 2.50312891113892 DPYSL3 dihydropyrimidinase-like 3 4.16840350145894 DPYSL4 dihydropyrimidinase-like 4 3.22268772155978 DSEL dermatan sulfate epimerase-like 2.51825736590280 DSP desmoplakin 2.27583067819754 DTWD1 DTW domain containing 1 2.16076058772688 ECM2 extracellular matrix protein 2, female organ and adipocyte specific 2.22222222222222 EDIL3 EGF-like repeats and discoidin I-like domains 3 3.50017500875044 EDNRA endothelin receptor type A 2.64970853206147 EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1 10.1010101010101 EFHD1 EF-hand domain family, member D1 2.45278390973755 EGFLAM EGF-like, fibronectin type III and laminin G domains 4.26075841499787 epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) EGFR oncogene homolog, avian) 2.87026406429392 EGR1 early growth response 1 3.55113636363636 EHBP1 EH domain binding protein 1 2.255299954894 EHD2 EH-domain containing 2 3.15756236185665 ELOVL family member 6, elongation of long chain fatty acids (FEN1/Elo2, ELOVL6 SUR4/Elo3-like, yeast) 3.6941263391208 EML1 echinoderm microtubule associated protein like 1 3.88500388500388 EMX2 empty spiracles homeobox 2 3.47222222222222 ENO2 enolase 2 (gamma, neuronal) 3.29924117452986 EPDR1 ependymin related protein 1 (zebrafish) 3.83877159309021 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, ERBB2 neuro/glioblastoma derived oncogene homolog (avian) 2.63643553915107 ERRFI1 ERBB receptor feedback inhibitor 1 2.62743037309511 ESM1 endothelial cell-specific molecule 1 5.85137507314219 EYA2 eyes absent homolog 2 (Drosophila) 2.35793444942231 FAM101A family with sequence similarity 101, member A 2.88600288600289 FAM114A1 family with sequence similarity 114, member A1 2.6816840976133 FAM155A family with sequence similarity 155, member A 2.38549618320611 FAM171A1 family with sequence similarity 171, member A1 2.97707651086633 FAM176A family with sequence similarity 176, member A 10.9529025191676 FAM180A family with sequence similarity 180, member A 3.68324125230203 FAP fibroblast activation protein, alpha 4.60617227084293 FAT1 FAT tumor suppressor homolog 1 (Drosophila) 8.75656742556918 FBLN1 fibulin 1 7.12250712250712 FBXL2 F-box and leucine-rich repeat protein 2 2.39865675221876 FBXO32 F-box protein 32 2.38891543239369 FERMT2 fermitin family homolog 2 (Drosophila) 3.38180588434224 FEZ1 fasciculation and elongation protein zeta 1 (zygin I) 3.61271676300578 FGF2 fibroblast growth factor 2 (basic) 2.25225225225225 FHL2 four and a half LIM domains 2 9.28505106778087 FKBP9L FK506 binding protein 9-like 3.58551452133381 FLG filaggrin 2.50563768479078 FLJ39632 hypothetical LOC642477 2.2456770716371 FLNC filamin C, gamma 11.8483412322275 FLRT3 fibronectin leucine rich transmembrane protein 3 2.39291696578129 FN1 fibronectin 1 3.09501702259362 FNDC1 fibronectin type III domain containing 1 4.04530744336570 FNDC1 fibronectin type III domain containing 1 6.28535512256443 FOLR3 folate receptor 3 (gamma) 3.01932367149758 FOSB FBJ murine osteosarcoma viral oncogene homolog B 3.19590923617769 FOXC1 forkhead box C1 3.295978905735 FOXD1 forkhead box D1 4.62534690101758 FRMD6 FERM domain containing 6 4.11692054343351 FST follistatin 2.33481204763017 FSTL1 follistatin-like 1 10.8813928182807 FSTL3 follistatin-like 3 (secreted glycoprotein) 2.80898876404494 FUT8 fucosyltransferase 8 (alpha (1,6) fucosyltransferase) 2.95333727111636 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- GALNT5 acetylgalactosaminyltransferase 5 (GalNAc-T5) 2.94031167303734 GAMT guanidinoacetate N-methyltransferase 2.76166804750069 GDF15 growth differentiation factor 15 2.91036088474971 GJA1 gap junction protein, alpha 1, 43kDa 23.3644859813084 GLIS3 GLIS family zinc finger 3 2.75938189845475 GNG11 guanine nucleotide binding protein (G protein), gamma 11 9.79431929480901 GNG12 guanine nucleotide binding protein (G protein), gamma 12 2.31803430690774 GPR1 G protein-coupled receptor 1 2.70416441319632 GPR177 G protein-coupled receptor 177 2.75406224180667 GPX7 glutathione peroxidase 7 4.76871721506915 GPX8 glutathione peroxidase 8 (putative) 12.0048019207683 GRB14 growth factor receptor-bound protein 14 2.19538968166850 HAS2 hyaluronan synthase 2 3.08071472581639 HBEGF heparin-binding EGF-like growth factor 3.1496062992126 HDDC2 HD domain containing 2 2.59740259740260 HECW2 HECT, C2 and WW domain containing E3 ubiquitin protein ligase 2 2.92056074766355 HEPH hephaestin 2.22568439795237 HOXA10 homeobox A10 2.56607646907878 HOXA5 homeobox A5 2.17533173809006 HOXB2 homeobox B2 2.12494687632809 HOXB7 homeobox B7 2.2737608003638 HOXC9 homeobox C9 2.72108843537415 HS3ST3A1 heparan sulfate (glucosamine) 3-O-sulfotransferase 3A1 3.68188512518409 HSPB6 heat shock protein, alpha-crystallin-related, B6 2.52206809583859 HTRA1 HtrA serine peptidase 1 19.7238658777120 ID3 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein 2.7063599458728 IGFBP2 insulin-like growth factor binding protein 2, 36kDa 3.79794910748196 IGFBP3 insulin-like growth factor binding protein 3 21.505376344086 IGFBP4 insulin-like growth factor binding protein 4 8.40336134453782 IGFBP6 insulin-like growth factor binding protein 6 13.0548302872063 IGFBP7 insulin-like growth factor binding protein 7 12.531328320802 IKIP IKK interacting protein 2.24315836698071 IQCK IQ motif containing K 2.14316330904415 IRS1 insulin receptor substrate 1 2.63019463440295 IRX3 iroquois homeobox 3 7.34753857457752 ISLR immunoglobulin superfamily containing leucine-rich repeat 4.82625482625483 ITGA11 integrin, alpha 11 4.21585160202361 ITGB5 integrin, beta 5 6.04960677555959 ITM2C integral membrane protein 2C 3.48189415041783 ITPR3 inositol 1,4,5-triphosphate receptor, type 3 10.3092783505155 KANK2 KN motif and ankyrin repeat domains 2 2.17060994139353 KCNK2 potassium channel, subfamily K, member 2 2.83848992336077 potassium large conductance calcium-activated channel, subfamily M, KCNMA1 alpha member 1 5.4585152838428 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor KDELR3 3 7.99360511590727 KIAA0101 KIAA0101 2.19538968166850 KIAA1199 KIAA1199 2.72034820457019 KIAA1217 KIAA1217 2.27324391907252 KIAA1644 KIAA1644 9.99000999001 KITLG KIT ligand 3.24254215304799 KLF2 Kruppel-like factor 2 (lung) 7.43494423791822 KLK3 kallikrein-related peptidase 3 3.10173697270471 KRT19 keratin 19 3.3955857385399 KRT34 keratin 34 3.6231884057971 KRT81 keratin 81 2.81769512538743 LAMA4 laminin, alpha 4 2.42718446601942 LAMB1 laminin, beta 1 2.94464075382803 LAPTM4B lysosomal protein transmembrane 4 beta 2.49376558603491 LARP6 La ribonucleoprotein domain family, member 6 3.57653791130186 LBH limb bud and heart development homolog (mouse) 2.46305418719212 LDOC1 leucine zipper, down-regulated in cancer 1 3.25945241199478 LEPR leptin receptor 2.58732212160414 LEPREL2 leprecan-like 2 5.51876379690949 LIF leukemia inhibitory factor (cholinergic differentiation factor) 4.04530744336570 LMCD1 LIM and cysteine-rich domains 1 2.59740259740260 LOX lysyl oxidase 19.53125 LOXL1 lysyl oxidase-like 1 3.26157860404436 LOXL4 lysyl oxidase-like 4 3.81533765738268 LPAR1 lysophosphatidic acid receptor 1 2.34192037470726 LPHN2 latrophilin 2 4.3010752688172 LUM lumican 12.7064803049555 MAGED1 melanoma antigen family D, 1 4.88042947779405 MALL mal, T-cell differentiation protein-like 3.74111485222596 MAP1B microtubule-associated protein 1B 3.24149108589951 MARCH4 membrane-associated ring finger (C3HC4) 4 2.84981476204047 MDK midkine (neurite growth-promoting factor 2) 2.30786983614124 MEG3 maternally expressed 3 (non-protein coding) 2.61028452101279 MEIS1 Meis homeobox 1 3.55492356914326 MEIS2 Meis homeobox 2 2.36127508854782 MFAP5 microfibrillar associated protein 5 3.07692307692308 MFGE8 milk fat globule-EGF factor 8 protein 11.5874855156431 MGC4294 hypothetical protein MGC4294 3.7243947858473 MGP matrix Gla protein 2.37925291458482 myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, MLLT11 Drosophila); translocated to, 11 2.70562770562771 MLPH melanophilin 4.11184210526316 MME membrane metallo-endopeptidase 5.75043128234618 matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV MMP2 collagenase) 2.29305205228159 MOCS1 molybdenum cofactor synthesis 1 3.26690623979092 MPDZ multiple PDZ domain protein 2.41721053903795 MPRIP myosin phosphatase Rho interacting protein 4.34216239687364 MRAP2 melanocortin 2 receptor accessory protein 2 3.23519896473633 MSRB2 methionine sulfoxide reductase B2 2.6567481402763 MSRB3 methionine sulfoxide reductase B3 3.15059861373661 MTMR11 myotubularin related protein 11 2.4588148512417 MXRA7 matrix-remodelling associated 7 7.85545954438335 MXRA8 matrix-remodelling associated 8 2.63157894736842 MXRA8 matrix-remodelling associated 8 4.1101520756268 MYH10 myosin, heavy chain 10, non-muscle 3.61663652802893 MYL6B myosin, light chain 6B, alkali, smooth muscle and non-muscle 2.51825736590280 MYL9 myosin, light chain 9, regulatory 2.62743037309511 MYLK myosin light chain kinase 2.60213374967473 MYO1B myosin IB 2.39234449760766 NAV3 neuron navigator 3 2.95770482105886 NCKAP1 NCK-associated protein 1 2.95945546019532 NDN necdin homolog (mouse) 5.44365813826892 NDUFA4L2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 3.4952813701503 NFIX nuclear factor I/X (CCAAT-binding transcription factor) 7.17360114777618 NGF nerve growth factor (beta polypeptide) 2.97000297000297 NGFRAP1 nerve growth factor receptor (TNFRSF16) associated protein 1 4.72813238770686 NHS Nance-Horan syndrome (congenital cataracts and dental anomalies) 2.30786983614124 NMB neuromedin B 2.64550264550265 NNMT nicotinamide N-methyltransferase 2.73224043715847 NOTCH3 Notch homolog 3 (Drosophila) 3.94632991318074 NR2F2 nuclear receptor subfamily 2, group F, member 2 5.18672199170125 NR2F6 nuclear receptor subfamily 2, group F, member 6 2.48447204968944 NRG1 neuregulin 1 2.9868578255675 NT5E 5'-nucleotidase, ecto (CD73) 6.92520775623269 NTM neurotrimin 4.73709142586452 NTN4 netrin 4 3.06748466257669 OCIAD2 OCIA domain containing 2 3.49406009783368 OLFML1 olfactomedin-like 1 2.18007412252017 OLFML3 olfactomedin-like 3 2.58197779499096 PALM paralemmin 3.46500346500346 PAMR1 peptidase domain containing associated with muscle regeneration 1 2.72108843537415 PAPPA pregnancy-associated plasma protein A, pappalysin 1 12.9198966408269 PAWR PRKC, apoptosis, WT1, regulator 3.21853878339234 PCDH18 protocadherin 18 3.03766707168894 PCGF2 polycomb group ring finger 2 2.12585034013605 PCOLCE procollagen C-endopeptidase enhancer 4.30477830391735 PCOLCE2 procollagen C-endopeptidase enhancer 2 2.45218244237371 PDE5A phosphodiesterase 5A, cGMP-specific 2.75027502750275 PDE7B phosphodiesterase 7B 2.60348867482426 PDGFRB platelet-derived growth factor receptor, beta polypeptide 10.6044538706257 PDZRN3 PDZ domain containing ring finger 3 2.20215811495265 PENK proenkephalin 8.48896434634974 PERP PERP, TP53 apoptosis effector 3.90777647518562 PFN2 profilin 2 5.60852495793606 PGF placental growth factor 3.46380325597506 PGM1 phosphoglucomutase 1 2.4390243902439 PGM2L1 phosphoglucomutase 2-like 1 2.36798484489699 PHGDH phosphoglycerate dehydrogenase 4.9825610363727 PHLDB2 pleckstrin homology-like domain, family B, member 2 2.60892251500130 PID1 phosphotyrosine interaction domain containing 1 2.40096038415366 PLAC9 placenta-specific 9 6.23052959501558 PLCG1 phospholipase C, gamma 1 2.47035573122530 pleckstrin homology domain containing, family A (phosphoinositide binding PLEKHA4 specific) member 4 2.32180171813327 PLOD1 procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1 2.45278390973755 PLOD2 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 13.1926121372032 PLS3 plastin 3 (T isoform) 3.79506641366224 PLS3 plastin 3 (T isoform) 6.6711140760507 POSTN periostin, osteoblast specific factor 7.11743772241993 PPAP2B phosphatidic acid phosphatase type 2B 2.35128144838937 PPAPDC3 phosphatidic acid phosphatase type 2 domain containing 3 2.34852043212776 PPP1R14C protein phosphatase 1, regulatory (inhibitor) subunit 14C 2.49128051818635 PPP1R3C protein phosphatase 1, regulatory (inhibitor) subunit 3C 5.93119810201661 PRG4 proteoglycan 4 3.43642611683849 PRKD1 protein kinase D1 3.82409177820268 PRRX1 paired related homeobox 1 11.3507377979569 PSG5 pregnancy specific beta-1-glycoprotein 5 2.9753049687593 PTGDS prostaglandin D2 synthase 21kDa (brain) 10.6496272630458 PTGES prostaglandin E synthase 10.989010989011 PTK2 PTK2 protein tyrosine kinase 2 2.38891543239369 PTPRK protein tyrosine phosphatase, receptor type, K 4.31965442764579 PTPRM protein tyrosine phosphatase, receptor type, M 2.34192037470726 PTRF polymerase I and transcript release factor 7.98084596967279 PVRL3 poliovirus receptor-related 3 2.91375291375291 PXDN peroxidasin homolog (Drosophila) 6.57462195923734 RAB11FIP5 RAB11 family interacting protein 5 (class I) 2.94031167303734 RAB23 RAB23, member RAS oncogene family 2.92056074766355 RAGE renal tumor antigen 3.74672161858374 RAI14 retinoic acid induced 14 5.96302921884317 RBM9 RNA binding motif protein 9 5.47645125958379 RCN3 reticulocalbin 3, EF-hand calcium binding domain 5.69476082004556 RECK reversion-inducing-cysteine-rich protein with kazal motifs 2.24719101123596 REXO2 REX2, RNA exonuclease 2 homolog (S. cerevisiae) 3.29055610398157 RGMB RGM domain family, member B 4.50450450450450 RGS4 regulator of G-protein signaling 4 10.4821802935010 RHOD ras homolog gene family, member D 2.55036980362153 RILPL1 Rab interacting lysosomal protein-like 1 2.20799293442261 RND3 Rho family GTPase 3 22.4215246636771 RPL29 ribosomal protein L29 2.20653133274492 RPS24 ribosomal protein S24 2.3696682464455 RRAD Ras-related associated with diabetes 2.30096640589047 RRAS2 related RAS viral (r-ras) oncogene homolog 2 3.385240352065 S100A13 S100 calcium binding protein A13 3.23939099449304 S100A16 S100 calcium binding protein A16 7.83699059561129 S1PR3 sphingosine-1-phosphate receptor 3 2.82885431400283 SATB2 SATB homeobox 2 2.59740259740260 SBSN suprabasin 2.35128144838937 SC65 synaptonemal complex protein SC65 2.90191526407429 SCARA3 scavenger receptor class A, member 3 2.28937728937729 SCG2 secretogranin II (chromogranin C) 2.11104074308634 SELM selenoprotein M 3.47584289190129 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), SERPINA3 member 3 8.57632933104631 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor SERPINE2 type 1), member 2 75.7575757575758 serpin peptidase inhibitor, clade H (heat shock protein 47), member 1, SERPINH1 (collagen binding protein 1) 3.78501135503407 SGCB sarcoglycan, beta (43kDa dystrophin-associated glycoprotein) 2.69614451334592 SGCE sarcoglycan, epsilon 4.90436488474742 SH3D19 SH3 domain containing 19 3.14762354422411 solute carrier family 1 (neuronal/epithelial high affinity glutamate SLC1A1 transporter, system Xag), member 1 2.40673886883273 solute carrier family 25 (mitochondrial carrier; adenine nucleotide SLC25A4 translocator), member 4 2.19876868953386 SLC39A14 solute carrier family 39 (zinc transporter), member 14 3.03766707168894 SLC44A2 solute carrier family 44, member 2 2.9086678301338 SLIT2 slit homolog 2 (Drosophila) 3.46020761245675 SWI/SNF related, matrix associated, actin dependent regulator of SMARCA1 chromatin, subfamily a, member 1 2.63435194942044 SNAI2 snail homolog 2 (Drosophila) 2.47586036147561 SNX7 sorting nexin 7 2.46852628980499 SOCS5 suppressor of cytokine signaling 5 2.37586124970302 SORBS2 sorbin and SH3 domain containing 2 3.35795836131632 SPOCK1 sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1 4.71253534401508 SPRY2 sprouty homolog 2 (Drosophila) 2.59470679813181 SRPX sushi-repeat-containing protein, X-linked 4.10677618069815 SRPX2 sushi-repeat-containing protein, X-linked 2 2.95420974889217 STC1 stanniocalcin 1 3.54233085370174 STC2 stanniocalcin 2 14.5348837209302 STEAP1 six transmembrane epithelial antigen of the prostate 1 3.7037037037037 SULF1 sulfatase 1 16.9779286926995 SYDE1 synapse defective 1, Rho GTPase, homolog 1 (C. elegans) 2.23914017017465 SYNC syncoilin, intermediate filament protein 2.32937339855579 TAGLN transgelin 15.1057401812689 TBC1D19 TBC1 domain family, member 19 3.05250305250305 TBX2 T-box 2 2.78164116828929 TCEA3 transcription elongation factor A (SII), 3 4.49640287769784 transcription factor AP-2 alpha (activating enhancer binding protein 2 TFAP2A alpha) 2.91800408520572

TFPI tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) 2.35349493998588 TFPI2 tissue factor pathway inhibitor 2 6.76132521974307 TGFB1I1 transforming growth factor beta 1 induced transcript 1 3.52858151023289 TGFB3 transforming growth factor, beta 3 2.24870699347875 TGFBR3 transforming growth factor, beta receptor III 3.46981263011797 THBS2 thrombospondin 2 5.47645125958379 THY1 Thy-1 cell surface antigen 8.70322019147084 TIMP1 TIMP metallopeptidase inhibitor 1 4.55788514129444 TIMP3 TIMP metallopeptidase inhibitor 3 3.34112930170398 TJP1 tight junction protein 1 (zona occludens 1) 2.18435998252512 TM4SF1 transmembrane 4 L six family member 1 11.6009280742459 TMEM119 transmembrane protein 119 4.97265042267529 TMEM158 transmembrane protein 158 2.67094017094017 TMEM171 transmembrane protein 171 2.53613999492772 TMEM183B transmembrane protein 183B 0.249525900788502 TMEM200A transmembrane protein 200A 4.12371134020619 TMEM45A transmembrane protein 45A 2.32828870779977 TMEM47 transmembrane protein 47 6.34920634920635 TMEM99 transmembrane protein 99 2.32666356444858 TNFRSF12A tumor necrosis factor receptor superfamily, member 12A 4.26075841499787 TPM1 tropomyosin 1 (alpha) 11.2866817155756 TPM2 tropomyosin 2 (beta) 22.3214285714286 TPRG1L tumor protein p63 regulated 1-like 2.23015165031222 TPST1 tyrosylprotein sulfotransferase 1 2.81690140845070 TRAM2 translocation associated membrane protein 2 2.91885580852306 TSC22D1 TSC22 domain family, member 1 5.76701268742791 TSIX XIST antisense RNA (non-protein coding) 2.30096640589047 TSPAN5 tetraspanin 5 2.59336099585062 TSPAN6 tetraspanin 6 2.35460324935248 TSPAN9 tetraspanin 9 2.44738130200685 TUBB3 tubulin, beta 3 3.16455696202532 TUSC3 tumor suppressor candidate 3 4.81927710843374 UAP1 UDP-N-acteylglucosamine pyrophosphorylase 1 3.08737264587836 UCHL1 ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) 2.20994475138122 UGDH UDP-glucose dehydrogenase 3.08071472581639 VASN vasorin 11.6550116550117 VCAM1 vascular cell adhesion molecule 1 16.0256410256410 VEGFC vascular endothelial growth factor C 4.97512437810945 WBP5 WW domain binding protein 5 3.51864883884588 WISP2 WNT1 inducible signaling pathway protein 2 5.3850296176629 WNT5B wingless-type MMTV integration site family, member 5B 3.41180484476288 WWC1 WW and C2 domain containing 1 2.17485863418878 XYLT2 xylosyltransferase II 2.40096038415366 YAP1 Yes-associated protein 1, 65kDa 7.45156482861401 YES1 v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 3.88953714507974 ZDHHC9 zinc finger, DHHC-type containing 9 2.4975024975025 ZNF462 zinc finger protein 462 2.21631205673759

DCs + MSCs LPS vs MSCs LPS

Gene Symbol Gene Name Fold Change ABCA6 ATP-binding cassette, sub-family A (ABC1), member 6 2.64480296217932 v-abl Abelson murine leukemia viral oncogene homolog 2 (arg, Abelson- ABL2 related gene) 2.80033604032484 ACOT7 acyl-CoA thioesterase 7 2.94985250737463 ACOX3 acyl-Coenzyme A oxidase 3, pristanoyl 2.57931390250193 ACP5 acid phosphatase 5, tartrate resistant 2.89351851851852 ACTA2 actin, alpha 2, smooth muscle, aorta 0.362450163102573 ADAM19 ADAM metallopeptidase domain 19 (meltrin beta) 3.02571860816944 ADAM8 ADAM metallopeptidase domain 8 3.36473755047106 ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 0.151476134934941 ADAP2 ArfGAP with dual PH domains 2 2.4906600249066 AGPAT9 1-acylglycerol-3-phosphate O-acyltransferase 9 3.41530054644809 AIF1 allograft inflammatory factor 1 2.41429261226461 AIM2 absent in melanoma 2 4.37254044599913 ALOX15 arachidonate 15-lipoxygenase 3.08641975308642 AMICA1 adhesion molecule, interacts with CXADR antigen 1 3.09214594928881 ANKFN1 ankyrin-repeat and fibronectin type III domain containing 1 0.370535052615977 APOBEC3A apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A 2.59470679813181 APOD apolipoprotein D 2.57532835436518 APOL4 apolipoprotein L, 4 0.36613942589338 ARAP2 ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2 2.66098988823842 ARHGAP25 Rho GTPase activating protein 25 2.72405339144647 ARHGAP4 Rho GTPase activating protein 4 2.48632521133764 ARMCX2 armadillo repeat containing, X-linked 2 0.374686200307243 ARRDC2 arrestin domain containing 2 0.328223980043982 BDKRB1 bradykinin receptor B1 0.338707492209728 BDNF brain-derived neurotrophic factor 0.288550323176362 BIRC3 baculoviral IAP repeat-containing 3 2.56871307474955 BRSK1 BR serine/threonine kinase 1 2.53742704897234 C10orf116 chromosome 10 open reading frame 116 0.374559892126751 C13orf18 chromosome 13 open reading frame 18 2.67165375367352 C13orf18 chromosome 13 open reading frame 18 2.52461499621308 C15orf48 chromosome 15 open reading frame 48 2.49438762783737 C1QA complement component 1, q subcomponent, A chain 2.90107339715695 C1QC complement component 1, q subcomponent, C chain 2.86944045911047 C5orf13 chromosome 5 open reading frame 13 0.360295442262655 C5orf20 chromosome 5 open reading frame 20 2.83446712018141 C6orf150 chromosome 6 open reading frame 150 2.43842965130456 CA11 carbonic anhydrase XI 3.03490136570561 CA12 carbonic anhydrase XII 0.361245574741709 CALCRL calcitonin receptor-like 3.87446726075165 CAMK1G calcium/calmodulin-dependent protein kinase IG 2.56213169356905 CBLB Cas-Br-M (murine) ecotropic retroviral transforming sequence b 0.375530436741898 CCL1 chemokine (C-C motif) ligand 1 3.58166189111748 CCL13 chemokine (C-C motif) ligand 13 1.57927984838913 CCL15 chemokine (C-C motif) ligand 15 4.12031314379893 CCL17 chemokine (C-C motif) ligand 17 2.88433804441881 CCL18 chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated) 1.77273533061514 CCL22 chemokine (C-C motif) ligand 22 2.40269101393561 CCL23 chemokine (C-C motif) ligand 23 2.44379276637341 CCL3 chemokine (C-C motif) ligand 3 3.81825124093165 CCL4L1 chemokine (C-C motif) ligand 4-like 1 2.50878073256397 CCL5 chemokine (C-C motif) ligand 5 4.08997955010225 CCNA1 cyclin A1 3.25945241199478 CCND1 cyclin D1 0.290951411114344 CCR7 chemokine (C-C motif) receptor 7 3.09214594928881 CD1C CD1c molecule 3.15258511979823 CD226 CD226 molecule 2.58732212160414 CD248 CD248 molecule, endosialin 0.384511862190949 CD274 CD274 molecule 2.61233019853710 CD36 CD36 molecule (thrombospondin receptor) 2.84575981787137 CD38 CD38 molecule 3.10945273631841 CD40 CD40 molecule, TNF receptor superfamily member 5 3.03214069132808 CD53 CD53 molecule 2.59807742270720 CD69 CD69 molecule 3.06654400490647 CD70 CD70 molecule 2.51825736590280 CD74 CD74 molecule, major histocompatibility complex, class II invariant chain 3.05250305250305 CD80 CD80 molecule 2.85062713797035 CD83 CD83 molecule 2.93427230046948 CD86 CD86 molecule 2.90107339715695 CFB complement factor B 3.26797385620915 CHI3L2 chitinase 3-like 2 2.72553829381303 CLDN1 claudin 1 5.69476082004556 CLEC10A C-type lectin domain family 10, member A 2.98151460942159 CLEC5A C-type lectin domain family 5, member A 2.57201646090535 CLEC7A C-type lectin domain family 7, member A 2.60010400416017 CLECL1 C-type lectin-like 1 2.78009452321379 CLIP3 CAP-GLY domain containing linker protein 3 0.383259236547601 CMPK2 cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial 3.03951367781155 COL1A2 collagen, type I, alpha 2 0.370274373310623 COL6A3 collagen, type VI, alpha 3 0.348906179128432 COMP cartilage oligomeric matrix protein 0.392634182731949 COX7A1 cytochrome c oxidase subunit VIIa polypeptide 1 (muscle) 0.342055755088079 CREB5 cAMP responsive element binding protein 5 2.75027502750275 CRLF2 cytokine receptor-like factor 2 4.74158368895211 CRYAB crystallin, alpha B 0.307749122915000 CST7 cystatin F (leukocystatin) 4.86381322957198 CTGF connective tissue growth factor 0.186936852731147 CTSH cathepsin H 3.08737264587836 CXCL10 chemokine (C-X-C motif) ligand 10 3.07219662058372 CXCL9 chemokine (C-X-C motif) ligand 9 3.98724082934609 CXCR4 chemokine (C-X-C motif) receptor 4 2.460024600246 CXCR4 chemokine (C-X-C motif) receptor 4 2.57135510413988 CXCR7 chemokine (C-X-C motif) receptor 7 0.350938761186173 CYGB cytoglobin 0.288983932493353 CYP27B1 cytochrome P450, family 27, subfamily B, polypeptide 1 2.98864315600717 CYP4X1 cytochrome P450, family 4, subfamily X, polypeptide 1 0.356824264049955 CYR61 cysteine-rich, angiogenic inducer, 61 0.125648661213515 CYTIP cytohesin 1 interacting protein 2.44618395303327 DACT1 dapper, antagonist of beta-catenin, homolog 1 (Xenopus laevis) 0.372633775525414 DAPP1 dual adaptor of phosphotyrosine and 3-phosphoinositides 2.59201658890617 DDAH1 dimethylarginine dimethylaminohydrolase 1 0.319733981327535 DDX58 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 2.55493101686254 DDX60 DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 3.47222222222222 DHRS2 dehydrogenase/reductase (SDR family) member 2 2.93685756240822 DHX58 DEXH (Asp-Glu-X-His) box polypeptide 58 3.10173697270471 DKFZP564O0823 prostatic androgen-repressed message-1 2.85143997718848 DKFZP586I1420 hypothetical protein DKFZp586I1420 2.80977802753582 DKK1 dickkopf homolog 1 (Xenopus laevis) 0.150529865125241 DKK3 dickkopf homolog 3 (Xenopus laevis) 0.380459595190991 DLX5 distal-less homeobox 5 0.272457292319429 DNASE1L3 deoxyribonuclease I-like 3 3.03306035790112 DRD2 dopamine receptor D2 0.38287770885979 DUSP2 dual specificity phosphatase 2 2.75482093663912 DUSP5 dual specificity phosphatase 5 2.47463499133878 EFNA1 ephrin-A1 2.84900284900285 ELMO1 engulfment and cell motility 1 2.95595625184747 ENOX1 ecto-NOX disulfide-thiol exchanger 1 0.338134848177453 ENTPD1 ectonucleoside triphosphate diphosphohydrolase 1 3.02388872089507 ENTPD8 ectonucleoside triphosphate diphosphohydrolase 8 0.35253472467038 EOMES eomesodermin homolog (Xenopus laevis) 0.354132728946809 EPB41L3 erythrocyte membrane protein band 4.1-like 3 2.54971953085161 ESM1 endothelial cell-specific molecule 1 0.321471051531810 ETV3 ets variant 3 3.10945273631841 FAM128A family with sequence similarity 128, member A 0.322591051324236 FAM180A family with sequence similarity 180, member A 0.337883497769969 FAM186B family with sequence similarity 186, member B 2.74499039253363 FAM43A family with sequence similarity 43, member A 0.312139089178138 FAM49A family with sequence similarity 49, member A 2.98062593144560 FAT1 FAT tumor suppressor homolog 1 (Drosophila) 0.356341089691052 FBXO32 F-box protein 32 0.293160564040925 FCER1A Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide 3.48189415041783 FCER1G Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide 2.38606537819136 FCER2 Fc fragment of IgE, low affinity II, receptor for (CD23) 3.3500837520938 FCGR2A Fc fragment of IgG, low affinity IIa, receptor (CD32) 2.28206298493838 FERMT2 fermitin family homolog 2 (Drosophila) 0.357653791130186 FGF2 fibroblast growth factor 2 (basic) 0.353706847764573 FGL2 fibrinogen-like 2 2.65322366675511 FHL2 four and a half LIM domains 2 0.250312891113892 FKBP5 FK506 binding protein 5 2.42072137496974 FLG filaggrin 0.334974709409440 FLNC filamin C, gamma 0.311604138102954 FNDC1 fibronectin type III domain containing 1 0.384689363339104 FOXD1 forkhead box D1 0.361323890735655 FRMD6 FERM domain containing 6 0.316465710940220 FSD1L fibronectin type III and SPRY domain containing 1-like 2.72405339144647 FST follistatin 0.337120318241580 GAS1 growth arrest-specific 1 0.271739130434783 GBP4 guanylate binding protein 4 2.91800408520572 GBP5 guanylate binding protein 5 3.81825124093165 GEM GTP binding protein overexpressed in skeletal muscle 0.379420245864319 GJA1 gap junction protein, alpha 1, 43kDa 0.395303790963355 GJB2 gap junction protein, beta 2, 26kDa 2.4612355402412 GK glycerol kinase 2.57267815796244 GPR132 G protein-coupled receptor 132 3.48675034867503 GPR141 G protein-coupled receptor 141 2.51889168765743 GPR157 G protein-coupled receptor 157 2.42248062015504 GPR183 G protein-coupled receptor 183 2.54065040650406 GPX8 glutathione peroxidase 8 (putative) 0.380575430050236 GRP1 (general receptor for phosphoinositides 1)-associated scaffold GRASP protein 2.64410364886304 HBA1 hemoglobin, alpha 1 0.306447658739887 HBEGF heparin-binding EGF-like growth factor 0.331345261762757 HERC6 hect domain and RLD 6 2.59268861809697 HES4 hairy and enhancer of split 4 (Drosophila) 2.79955207166853 HESX1 HESX homeobox 1 4.38212094653812 HLA-DMB major histocompatibility complex, class II, DM beta 2.50187640730548 HLA-DPA1 major histocompatibility complex, class II, DP alpha 1 3.02755071147442 HLA-DPB1 major histocompatibility complex, class II, DP beta 1 2.73074822501365 HLA-DRB3 major histocompatibility complex, class II, DR beta 3 4.81927710843374 HLA-DRB4 major histocompatibility complex, class II, DR beta 4 2.66808964781217 HLA-DRB5 major histocompatibility complex, class II, DR beta 5 3.98565165404544 HLA-F major histocompatibility complex, class I, F 2.59605399792316 HOXA5 homeobox A5 0.396212211260351 HOXB8 homeobox B8 0.327193011157282 HTRA1 HtrA serine peptidase 1 0.391604010025063 ID1 inhibitor of DNA binding 1, dominant negative helix-loop-helix protein 0.252276798102878 ID3 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein 0.152564611112806 ID4 inhibitor of DNA binding 4, dominant negative helix-loop-helix protein 0.368976459301897 IDO1 indoleamine 2,3-dioxygenase 1 2.86450873675165 IFI44L interferon-induced protein 44-like 2.76243093922652 IFIT3 interferon-induced protein with tetratricopeptide repeats 3 2.4515812699191 IGFBP4 insulin-like growth factor binding protein 4 0.361271676300578 IGFBP7 insulin-like growth factor binding protein 7 0.35202590910691 IL10RA interleukin 10 receptor, alpha 4.22832980972516 IL15RA interleukin 15 receptor, alpha 2.65181649429859 IL18BP interleukin 18 binding protein 2.29095074455899 IL1R2 interleukin 1 receptor, type II 2.87686996547756 IL1RAP interleukin 1 receptor accessory protein 3.71885459278542 IL1RN interleukin 1 receptor antagonist 2.77238702522872 IL27 interleukin 27 4.00320256204964 IL2RA interleukin 2 receptor, alpha 2.81610813855252 IL4I1 interleukin 4 induced 1 3.58808754933620 IQCG IQ motif containing G 2.44081034903588 IRF2BP2 interferon regulatory factor 2 binding protein 2 0.368039453829451 IRF4 interferon regulatory factor 4 2.48508946322068 IRF7 interferon regulatory factor 7 3.54107648725212 IRS1 insulin receptor substrate 1 0.376818147561987 IRX3 iroquois homeobox 3 0.178903678259625 IRX5 iroquois homeobox 5 0.313361744798195 ISG15 ISG15 ubiquitin-like modifier 3.11429461227032 ISG20 interferon stimulated exonuclease gene 20kDa 3.66032210834553 ISLR immunoglobulin superfamily containing leucine-rich repeat 0.385030032342523 ITGAX integrin, alpha X (complement component 3 receptor 4 subunit) 2.93513354857646 ITGB5 integrin, beta 5 0.390548720952939 ITPR3 inositol 1,4,5-triphosphate receptor, type 3 0.375544539582394 ITPRIP inositol 1,4,5-triphosphate receptor interacting protein 0.335874785879824 JAK2 Janus kinase 2 2.77932184546971 KBTBD6 kelch repeat and BTB (POZ) domain containing 6 0.342219636562746 KCNE1 potassium voltage-gated channel, Isk-related family, member 1 2.87356321839080 KIAA0101 KIAA0101 0.350336322869955 KIAA1199 KIAA1199 0.366152832192157 KIAA1644 KIAA1644 0.355113636363636 KRT17 keratin 17 3.10077519379845 KRT34 keratin 34 0.242913012850098 KRTAP2-1 keratin associated protein 2-1 0.373999551200539 KYNU kynureninase (L-kynurenine hydrolase) 2.55232261357836 LAD1 ladinin 1 3.04228780042592 LAMB2 laminin, beta 2 (laminin S) 0.389044506691566 LAMP3 lysosomal-associated membrane protein 3 3.49040139616056 LAPTM5 lysosomal protein transmembrane 5 2.63574064312072 LCP1 lymphocyte cytosolic protein 1 (L-plastin) 2.96208530805687 LGALS9 lectin, galactoside-binding, soluble, 9 2.45398773006135 LGALS9C lectin, galactoside-binding, soluble, 9C 2.4588148512417 LGR6 leucine-rich repeat-containing G protein-coupled receptor 6 0.381562881562882 LIF leukemia inhibitory factor (cholinergic differentiation factor) 0.358744394618834 leukocyte immunoglobulin-like receptor, subfamily A (without TM domain), LILRA3 member 3 2.43546030199708 LOC100133678 similar to hCG2042724 3.55618776671408 LOC401433 hypothetical LOC401433 2.52908447142135 LOC401442 hypothetical LOC401442 0.307588200916613 LOR loricrin 3.47342827370615 LPAR6 lysophosphatidic acid receptor 6 2.73597811217510 LRRC50 leucine rich repeat containing 50 3.34224598930481 LRRFIP2 leucine rich repeat (in FLII) interacting protein 2 2.47463499133878 LST1 leukocyte specific transcript 1 2.47708694575180 LY86 lymphocyte antigen 86 2.58665287118469 LYN v-yes-1 Yamaguchi sarcoma viral related oncogene homolog 2.50689395838556 LYPD4 LY6/PLAUR domain containing 4 3.09119010819165 LYSMD2 LysM, putative peptidoglycan-binding, domain containing 2 2.44081034903588 MALL mal, T-cell differentiation protein-like 0.376619463693884 MCOLN2 mucolipin 2 2.85062713797035 MFGE8 milk fat globule-EGF factor 8 protein 0.382306839469358 MGC4294 hypothetical protein MGC4294 0.325182101977107 MMP12 matrix metallopeptidase 12 (macrophage elastase) 3.87897595034911 MMP3 matrix metallopeptidase 3 (stromelysin 1, progelatinase) 0.331575980635963 matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV MMP9 collagenase) 2.23964165733483 MNDA myeloid cell nuclear differentiation antigen 2.64970853206147 MS4A7 membrane-spanning 4-domains, subfamily A, member 7 2.86368843069874 MT1E metallothionein 1E 0.384585801092224 MTMR8 myotubularin related protein 8 0.312597686777118 MTSS1 metastasis suppressor 1 0.376236878738854 myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 MX1 (mouse) 2.76319425255595 MX2 myxovirus (influenza virus) resistance 2 (mouse) 3.48796651552145 MXD1 MAX dimerization protein 1 2.70489586150933 v-myc myelocytomatosis viral related oncogene, neuroblastoma derived MYCN (avian) 0.271584150348986 MYH10 myosin, heavy chain 10, non-muscle 0.356125356125356 MYO1G myosin IG 2.75103163686382 NAMPT nicotinamide phosphoribosyltransferase 2.51004016064257 NCF1 neutrophil cytosolic factor 1 2.87935502447452 NCF1C neutrophil cytosolic factor 1C pseudogene 2.82725473565168 NDP Norrie disease (pseudoglioma) 3.29380764163373 NDRG2 NDRG family member 2 2.71076172404446 NEDD9 neural precursor cell expressed, developmentally down-regulated 9 0.329706561160567 NGFRAP1 nerve growth factor receptor (TNFRSF16) associated protein 1 0.355075808685154 NIACR1 niacin receptor 1 4.38788942518649 NIACR2 niacin receptor 2 3.89256520046711 NR4A2 nuclear receptor subfamily 4, group A, member 2 0.348334958896475 NR4A3 nuclear receptor subfamily 4, group A, member 3 2.80269058295964 NT5C3 5'-nucleotidase, cytosolic III 2.88267512251369 NT5C3 5'-nucleotidase, cytosolic III 2.78473962684489 NT5DC2 5'-nucleotidase domain containing 2 0.37397157816006 OAS2 2'-5'-oligoadenylate synthetase 2, 69/71kDa 3.27868852459016 OASL 2'-5'-oligoadenylate synthetase-like 3.67242012486228 OSCAR osteoclast associated, immunoglobulin-like receptor 2.97885016383676 PAQR6 progestin and adipoQ receptor family member VI 0.312412134087288 PARP10 poly (ADP-ribose) polymerase family, member 10 3.36473755047106 PARP14 poly (ADP-ribose) polymerase family, member 14 3.01659125188537 PCTK3 PCTAIRE protein kinase 3 0.362923713435436 PDGFRL platelet-derived growth factor receptor-like 3.30250990752972 PELI1 pellino homolog 1 (Drosophila) 3.46500346500346 PENK proenkephalin 0.296735905044510 PGK2 phosphoglycerate kinase 2 0.420185722089163 PHLDA1 pleckstrin homology-like domain, family A, member 1 0.300426605780208 PI4K2B phosphatidylinositol 4-kinase type 2 beta 2.43486729973216 PILRA paired immunoglobin-like type 2 receptor alpha 2.4588148512417 PKIB protein kinase (cAMP-dependent, catalytic) inhibitor beta 3.07314074984634 PLAGL2 pleiomorphic adenoma gene-like 2 2.59403372243839 PLAT plasminogen activator, tissue 2.84414106939704 PLCXD1 phosphatidylinositol-specific phospholipase C, X domain containing 1 0.37913254473764 PLEK pleckstrin 2.51762336354481 PNPLA5 patatin-like phospholipase domain containing 5 3.51493848857645 PPM1K protein phosphatase 1K (PP2C domain containing) 2.92483182217023 PPP1R3C protein phosphatase 1, regulatory (inhibitor) subunit 3C 0.348371363873890 PRAGMIN homolog of rat pragma of Rnd2 2.61985852763951 PTGDS prostaglandin D2 synthase 21kDa (brain) 0.370727367094239 PTGER3 prostaglandin E receptor 3 (subtype EP3) 2.81452293836195 PTPRO protein tyrosine phosphatase, receptor type, O 2.69323996768112 RAB11FIP1 RAB11 family interacting protein 1 (class I) 2.47524752475248 RAB23 RAB23, member RAS oncogene family 0.382995021064726 RAB30 RAB30, member RAS oncogene family 2.43724104313917 RAGE renal tumor antigen 0.320769847634322 RAP1GAP RAP1 GTPase activating protein 2.74122807017544 RASD1 RAS, dexamethasone-induced 1 0.30507337014552 RASGRP3 RAS guanyl releasing protein 3 (calcium and DAG-regulated) 2.50187640730548 RASSF2 Ras association (RalGDS/AF-6) domain family member 2 2.4740227610094 RASSF4 Ras association (RalGDS/AF-6) domain family member 4 2.43131534159981 RDM1 RAD52 motif 1 0.388485295831553 REL v-rel reticuloendotheliosis viral oncogene homolog (avian) 2.40500240500241 RELT RELT tumor necrosis factor receptor 0.308794466403162 RGS1 regulator of G-protein signaling 1 3.10655483069276 RGS12 regulator of G-protein signaling 12 2.18818380743982 RNASE1 ribonuclease, RNase A family, 1 (pancreatic) 2.06440957886045 RNASE6 ribonuclease, RNase A family, k6 3.60100828231905 RNF144B ring finger protein 144B 2.41312741312741 RNF19A ring finger protein 19A 2.71517784414879 RNF19B ring finger protein 19B 2.81452293836195 RSAD2 radical S-adenosyl methionine domain containing 2 2.96296296296296 RTP4 receptor (chemosensory) transporter protein 4 2.63713080168776 RUNX3 runt-related transcription factor 3 2.62536098713573 S100A13 S100 calcium binding protein A13 0.390502967822555 S100A16 S100 calcium binding protein A16 0.330797221303341 S100A4 S100 calcium binding protein A4 3.35345405767941 SAMSN1 SAM domain, SH3 domain and nuclear localization signals 1 2.93083235638921 sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) SEMA4A and short cytoplasmic domain, (semaphorin) 4A 4.05022276225192 sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) SEMA4D and short cytoplasmic domain, (semaphorin) 4D 3.03490136570561 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), SERPINA1 member 1 3.01295570955107 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor SERPINE1 type 1), member 1 0.371319297463889 SGK1 serum/glucocorticoid regulated kinase 1 0.292517404785585 SGPP2 sphingosine-1-phosphate phosphotase 2 3.62187613183629 SH3BP4 SH3-domain binding protein 4 0.328795949233905 SH3RF1 SH3 domain containing ring finger 1 0.358435786228897 SIGLEC1 sialic acid binding Ig-like lectin 1, sialoadhesin 2.79955207166853 SLAMF7 SLAM family member 7 2.53742704897234 SLC16A10 solute carrier family 16, member 10 (aromatic amino acid transporter) 2.81690140845070

SLC1A3 solute carrier family 1 (glial high affinity glutamate transporter), member 3 2.50375563345018

solute carrier family 25 (mitochondrial carrier; adenine nucleotide SLC25A4 translocator), member 4 0.377116566730776 solute carrier family 7, (cationic amino acid transporter, y+ system) member SLC7A11 11 2.3719165085389 SMAP2 small ArfGAP2 2.61437908496732 SOCS3 suppressor of cytokine signaling 3 2.62398320650748 SORBS1 sorbin and SH3 domain containing 1 3.69685767097967 SP110 SP110 nuclear body protein 2.53549695740365 SP140 SP140 nuclear body protein 3.09310238168883 SPI1 spleen focus forming virus (SFFV) proviral integration oncogene spi1 2.60416666666667 SPINT2 serine peptidase inhibitor, Kunitz type, 2 2.56541816316060 SPRY2 sprouty homolog 2 (Drosophila) 0.220375961390132 SRGAP2 SLIT-ROBO Rho GTPase activating protein 2 2.66453503863576 ST8SIA4 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4 2.8184892897407 STAB1 stabilin 1 2.67308206361935 STARD5 StAR-related lipid transfer (START) domain containing 5 2.73747604708459 STC2 stanniocalcin 2 0.384009830651665 STK4 serine/threonine kinase 4 2.79017857142857 STX11 syntaxin 11 2.65251989389920 SYNC syncoilin, intermediate filament protein 0.328914909712857 TACSTD2 tumor-associated calcium signal transducer 2 3.55998576005696 TAGLN transgelin 0.416146483562214 TRAF-interacting protein with forkhead-associated domain, family member TIFAB B 2.93513354857646 TIMP3 TIMP metallopeptidase inhibitor 3 0.361899247249566 TLR8 toll-like receptor 8 2.84656988329063 TMEM119 transmembrane protein 119 0.395741818037912 TMEM123 transmembrane protein 123 2.53292806484296 TMEM149 transmembrane protein 149 2.64131008980454 TMEM183B transmembrane protein 183B 0.174910795494298 TMEM194A transmembrane protein 194A 3.15756236185665 TMEM51 transmembrane protein 51 2.44439012466390 TNFRSF12A tumor necrosis factor receptor superfamily, member 12A 0.323897130271426 TNFRSF4 tumor necrosis factor receptor superfamily, member 4 2.50752256770311 TNFRSF6B tumor necrosis factor receptor superfamily, member 6b, decoy 3.38180588434224 TNFRSF9 tumor necrosis factor receptor superfamily, member 9 3.36813742000674 TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 3.57653791130186 TNFSF13B tumor necrosis factor (ligand) superfamily, member 13b 3.11817898347365 TPM1 tropomyosin 1 (alpha) 0.247218788627936 TPM2 tropomyosin 2 (beta) 0.286902883373978 TPRX1 tetra-peptide repeat homeobox 1 2.64620269912675 TRIM15 tripartite motif-containing 15 2.95072292711714 TSC22D1 TSC22 domain family, member 1 0.304506699147381 TTC39B tetratricopeptide repeat domain 39B 2.51825736590280 TTYH2 tweety homolog 2 (Drosophila) 3.44471236651740 TYMP thymidine phosphorylase 2.65181649429859 UNC93B1 unc-93 homolog B1 (C. elegans) 2.49003984063745 USP41 ubiquitin specific peptidase 41 2.54582484725051 VAC14 Vac14 homolog (S. cerevisiae) 2.71149674620390 VASN vasorin 0.372286958787834 VEGFC vascular endothelial growth factor C 0.384172109104879 WARS tryptophanyl-tRNA synthetase 2.88350634371396 WARS tryptophanyl-tRNA synthetase 2.87273771904625 WNT5B wingless-type MMTV integration site family, member 5B 0.387431715160203 XAF1 XIAP associated factor 1 2.77392510402219 ZC3HAV1 zinc finger CCCH-type, antiviral 1 3.61271676300578 ZNF366 zinc finger protein 366 2.77623542476402