TLR1/2 Ligand−Stimulated Mouse Liver Endothelial Cells Secrete IL-12 and Trigger CD8+ T Cell Immunity In Vitro

This information is current as Jia Liu, Min Jiang, Zhiyong Ma, Kirsten K. Dietze, of October 2, 2021. Gennadiy Zelinskyy, Dongliang Yang, Ulf Dittmer, Joerg F. Schlaak, Michael Roggendorf and Mengji Lu J Immunol published online 13 November 2013 http://www.jimmunol.org/content/early/2013/11/12/jimmun ol.1301262 Downloaded from

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 13, 2013, doi:10.4049/jimmunol.1301262 The Journal of Immunology

TLR1/2 Ligand–Stimulated Mouse Liver Endothelial Cells Secrete IL-12 and Trigger CD8+ T Cell Immunity In Vitro

Jia Liu,* Min Jiang,† Zhiyong Ma,* Kirsten K. Dietze,* Gennadiy Zelinskyy,* Dongliang Yang,‡ Ulf Dittmer,* Joerg F. Schlaak,† Michael Roggendorf,* and Mengji Lu*

Liver sinusoidal endothelial cells (LSECs) are unique organ-resident APCs capable of Ag cross-presentation and subsequent tol- erization of naive CD8+ T cells. Under certain conditions, LSECs can switch from a tolerogenic to an immunogenic state and promote the development of T cell immunity. However, little is known about the mechanisms of LSECs to induce T cell immunity. In this study, we investigated whether functional maturation of LSECs can be achieved by TLR ligand stimulation and elucidated the mechanisms involved in LSEC-induced T cell immunity. We demonstrate that pretreatment of LSECs with palmitoyl-3-

cysteine-serine-lysine-4 (P3C; TLR1/2 ligand) but not poly(I:C) (TLR3 ligand) or LPS (TLR4 ligand) reverted their suppressive Downloaded from properties to induce T cell immunity. Importantly, P3C stimulation caused functional maturation of Ag-presenting LSECs and enabled them to activate virus-specific CD8+ T cells. The LSEC-mediated CD8+ T cell immunity was initiated by soluble mediators, one of which was IL-12 secreted at a low but sustained level after P3C stimulation. P3C stimulation did not induce programmed death ligand 1 expression on LSECs, thereby favoring T cell proliferation and activation instead of suppression. Our data suggest that LSECs undergo maturation exclusively in response to TLR1/2 ligand stimulation and that the immunological status of LSECs

was dependent upon the balance between programmed death ligand 1 and IL-12 expression. These results have implications for our http://www.jimmunol.org/ understanding of liver-specific tolerance and autoimmunity and for the development of strategies to overcome T cell tolerance in situations such as chronic viral liver infections or liver cancer. The Journal of Immunology, 2013, 191: 000–000.

oll-like receptors, as evolutionarily conserved, germline- The liver is continuously exposed to food and microbial Ags that encoded pattern recognition receptors (PRRs), play a cru- are transported from the gut via the portal circulation, and it T cial role in early host defense by recognizing so-called exhibits barrier functions toward environmental Ags. Therefore, pathogen-associated molecular patterns (PAMP). Thus, they serve the risk of immune activation in the liver appears quite high in the as an important link between innate and adaptive immunity (1). body. It seems that the liver has in turn acquired special mecha- by guest on October 2, 2021 Ligation of TLRs or cytoplasmic PRRs causes functional matura- nisms of immune tolerance to avoid an overactivation of innate and tion of professional APCs, in particular dendritic cells (DCs) (2). adaptive immune responses (8, 9). The liver is known for its ability This leads to increased expression of costimulatory molecules such to induce Ag-specific immune tolerance rather than immunity as CD80/CD86 and CD40 as well as increased release of IL-12, (10). Liver nonparenchymal cells, including liver sinusoidal en- which are required to promote cytotoxic CD8+ T cell differentia- dothelial cells (LSECs) and Kupffer cells, play a crucial role in tion and activation (3–5). maintaining the homeostasis of the hepatic microenvironment Previously, TLR2 was able to mediate antiviral actions against through induction of Ag-specific T cell tolerance (11–13). Among hepatitis B virus (HBV) and was showntoplayanimportantrolein those different cell populations involved in the induction of he- HBV infection (6). Our previous experiments indicated that TLR2 patic tolerance, LSECs are particularly important because they are may activate various APCs, including liver nonparenchymal cells (7). strategically located in the liver sinusoids to interact with pas- senger leukocytes (14). In addition, LSECs have extraordinary *Institute of Virology, University Hospital of Essen, University of Duisburg-Essen, scavenger function. For example, LSECs take up more hepato- 45122 Essen, Germany; †Department of Gastroenterology and Hepatology, Univer- tropic viruses (such as HBV and hepatitis C virus) from the blood sity Hospital of Essen, University of Duisburg-Essen, 45122 Essen, Germany; and ‡ than other hepatic cells during viral infection (15, 16), and they Department of Infectious Diseases, Union Hospital, Tongji Medical College, + + Huazhong University of Science and Technology, 430022 Wuhan, China are highly efficient in presenting Ags to CD4 and CD8 T cells. + Received for publication May 13, 2013. Accepted for publication October 12, 2013. However, cross-presentation to CD8 T cells by LSECs can in- duce Ag-specific immune tolerance in contrast to professional This work was supported by Deutsche Forschungsgemeinschaft Grants Transregio + TRR60 and GRK1045/2. APCs such as DCs (11). Naive CD8 T cells that are primed by Address correspondence and reprint requests to Prof. Mengji Lu, Institute of Virol- LSECs are initially activated to proliferate and express activation ogy, University Hospital of Essen, Hufelandstrasse 55, 45122 Essen, Germany. markers, like CD69 and CD25, but finally exhibit low IL-2 and E-mail address: [email protected] IFN-g production and low cytotoxicity (11). The induction of The online version of this article contains supplemental material. tolerance correlates with the expression of the negative costimu- Abbreviations used in this article: bmDC, bone marrow–derived dendritic cell; DC, latory molecule ligand programmed death ligand 1 (PD-L1) by dendritic cell; FDR, false discovery rate; FV, Friend virus; HBV, hepatitis B virus; LSEC, liver sinusoidal endothelial cell; P3C, palmitoyl-3-cysteine-serine-lysine-4; LSECs, as LSECs from PD-L1–deficient mice failed to induce + PAMP, pathogen-associated molecular pattern; PD-1, programmed death 1; PD-L1, CD8 T cell tolerance (13). programmed death ligand 1; PRR, pattern recognition receptor; tg, transgenic; However, it is still unclear whether ligation of TLRs or PRRs can WHcAg, woodchuck hepatitis virus core Ag. cause functional maturation of LSECs and revert their suppressive Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 properties to induce T cell immunity. Although it has been reported

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301262 2 ACTIVATION OF LSECs BY TLR1/2 LIGANDS that murine CMV-infected LSECs were able to trigger CD8+ T cell analyzed for activation/proliferation by flow cytometry. For determination of the maturation of LSEC function, corresponding peptide-loaded LSECs or immunity, the authors failed to clarify the mechanisms of the + + LSEC maturation (14). Physiologic exposure of the liver to bac- DCs were cocultured with FV TCR tg CD8 T cells or CD8 Tcellsfrom woodchuck hepatitis virus core Ag (WHcAg)-immunized mice for either 3 terial degradation products is normally not associated with local or 7 d. Blocking Abs were added when indicated. After 3 d, cytokine con- inflammatory responses (17). This suggests that organ-resident APCs centrations in supernatants were assessed by ELISA. After 7 d, intracellular such as LSECs may have evolved the ability to differentiate vari- IFN-g staining was performed to determine the percentage of IFN-g–pro- + + eties of TLR stimulations, and thus perform different sentinel ducing CD8 T cells. The ratio of CD8 T cells to LSECs/DCs was 2:1. functions such as inducing immune tolerance or immunity. There- DNA vaccination fore, we determined whether organ-resident LSECs show plasticity Recombinant eukaryotic expression plasmid pCGWHc, which expresses and switch from a tolerogenic to an immunogenic APC upon PAMP WHcAg, was constructed by our laboratory (A. Kosinska, unpublished stimulation. In this study, we show that LSECs undergo immuno- data). Mice were pretreated with cardiotoxin (Latoxan, Valence, France) by logical maturation exclusively in response to TLR1/2 ligand stim- tibialis anterior muscle injection (10 mM in sterile PBS, 50 ml per limb) ulation, which results in generation of virus-specific CD8+ Tcell 7 d before the prime DNA vaccination. For each vaccination, 100 mg immunity. This maturation status of LSECs is achieved by regu- pCGWHc plasmid was injected to tibialis anterior muscle of each mouse. Prime-boost DNA vaccination was performed, and the interval of prime lating the balance between PD-L1 and IL-12 expression. and 23 boost DNA vaccination was 2 wk. Materials and Methods Quantitative RT-PCR Mice Total RNA was isolated from 1 3 106 to 10 3 106 cells using TRIzol (Life Technologies). One-step real-time RT-PCR was carried out with the QuantiTect Inbred C57BL/6 wild-type and DbGagL TCR transgenic (tg) mice were SYBR Green RT-PCR kit (Qiagen, Hilden, Germany) on the iCycler real-time Downloaded from maintained under pathogen-free conditions. The DbGagLTCR tg mice were amplification system (Bio-Rad, Hercules, CA), as described previously (21). on a C57BL/6 or B6.SJL (CD45.1 congenic) background, and .90% of the CD8+ T cells contained a TCR specific for the DbGagL Friend virus (FV) In vitro cytotoxicity assays epitope (FV-TCR CD8+ T cells) (18). All mice were females, 8–10 wk of age, and were kept in the Animal Care Center, University of Duisburg- The in vitro cytotoxicity assays were performed, as described earlier Essen (Essen, Germany). Experiments were conducted in accordance with (14). Briefly, EL4 cells were either loaded with FV peptide and labeled CFSEhigh (2 mmol/L), or remained unloaded labeled CFSElow (0.2 mmol/L) the Guide for the Care and Use of Laboratory Animals and were reviewed 3 3 http://www.jimmunol.org/ and approved by the local Animal Care and Use Committee (Animal Care as controls. Cells were mixed at a 1:1 ratio, and specific killing of 5 10 Center, University of Duisburg-Essen, Essen, Germany, and the district target cells in vitro was determined after 4 h. Specific kill was determined 2 3 government of Du¨sseldorf, Germany). using the following formula: percentage of specific kill = 100 (100 [CFSEhigh/CFSElow]sample/[CFSEhigh/CFSElow]control). Reagents and Abs FV infection and adoptive cell transfers Agonists for TLR1/2 (palmitoyl-3-cysteine-serine-lysine-4 [P3C]), TLR3 Acute FV infection was performed, as described previously (22). Adoptive (polyinosine-polycytidylic acid, poly I:C), and TLR4 (LPS from Escherichia cell transfers were done by i.v. injection of 0.5 ml PBBS containing 15 coli 011:B4 strain, LPS) were purchased from InvivoGen (San Diego, CA). U/ml heparin and 5 3 106 purified CD8+ T cells, as indicated at day 5 Mouse rIFN-g and rIL-2 were purchased from R&D Systems (Minneapolis, postinfection. All mice were sacrificed at day 8 postinfection. Spleens were MN). Neutralizing or blocking Abs anti–IL-12, anti–IL-23, and anti–PD-L1

taken for analysis of transferred cells by flow cytometry and for deter- by guest on October 2, 2021 were provided by eBioscience (San Diego, CA). mination of viral titers. Cell isolation Infectious center assays Isolation of LSECs, myeloid DCs, and CD8+ T cells was performed, as For the titrations of FV loads, single-cell suspensions were prepared with described previously (19). The purity of the cell fractions was monitored spleens from infected mice, plated onto susceptible Mus dunni cells (23), by flow cytometry and was .98% in all cases. Less than 0.5% of NK cells and cocultivated for 3 d. The cells were fixed with ethanol and stained with and DCs were found in the purified LSECs (Supplemental Fig. 1A). All Friend murine leukemia virus envelope-specific mAb 720 (24). The in- cell fractions contained ,5% dead cells after the separation procedure. fectious centers were visualized by using peroxidase-conjugated goat anti- Flow cytometry mouse Abs and 3-amino-9-ethylcarbazole for the detection of foci. Cell surface and intracellular staining for flow cytometry analysis was per- Bioinformatic analysis formed using BD Biosciences (Heidelberg, Germany) or eBioscience High throughput sequencing and data analysis were performed by BGI Hong (Frankfurt, Germany) reagents. Intracellular granzyme B and IFN-g staining Kong. Differentially expressed genes were selected using the following cri- was performed, as described (20). Data were acquired using a FACSCalibur teria: fold change .2 (log2 ratio $ 61), false discovery rate (FDR) ,0.001. flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR). Cell debris and dead cells were excluded from the Statistical analysis analysis based on scatter signals and 7-aminoactinomycin D fluorescence. Statistics comparing two groups were done using the nonparametric t test. T cell proliferation assay and cytokine assays When more than two groups were compared, a one-way ANOVA was used with a Tukey posttest (GraphPad Prism software; GraphPad, San Diego, CA). RBC-depleted splenocytes were cultured at 4 3 105 cells/well with or without LSECs in a total volume of 200 ml. Splenocytes were stimulated with 1 mg/ml anti-CD3 and 1 mg/ml anti-CD28. Cell-free supernatants Results were collected and subjected to assays to measure IFN-g, IL-2, and TNF-a Resident LSECs suppress T cell proliferation and cytokine production using cytokine ELISA kits (BD Biosciences). For the prolif- production via programmed death 1/PD-L1 interaction eration assay, 1 mCi/well [3H]thymidine (2 Ci/mM; PerkinElmer, Wood- bridge, Ontario, Canada) was added for the last 18 h. Cells were harvested, For the initial assessment of the immunosuppressive properties of and the incorporation of [3H]thymidine was measured using a liquid LSECs, T cells stimulated with anti-CD3 and anti-CD28 were scintillation counter (PerkinElmer, Rodgau, Germany). For cell contact cocultured with LSECs from C57BL/6 mice. The results indicated that studies, LSECs were added to the lower chamber of Transwell plates (0.4- LSECs potently suppressed T cell proliferation. In addition, activated mm pores, polycarbonate membrane; BD Labware, Heidelberg, Germany), and splenocytes were added to the upper chamber. T cells produced significantly less IFN-g,IL-2,andTNF-a in the presence of LSECs (Supplemental Fig. 1B). To distinguish whether + Analysis of CD8 T cell function the LSEC-mediated suppression of T cell functions is mediated by CFSE (Invitrogen, Darmstadt, Germany)-labeled FV TCR tg CD8+ Tcells a soluble factor or contact dependent, we assessed proliferation and were coincubated with LSECs or DCs in the presence of Ag for 3 d and cytokine production of activated T cells using a Transwell system. The Journal of Immunology 3

When LSECs and responder T cells were separated by a semiper- whether TLR stimulation can mediate maturation of LSECs, and meable membrane, suppression of T cell proliferation and cytokine hence abrogate the suppression of T cells by LSECs. T cells stim- production was abrogated (Supplemental Fig. 1C), suggesting that ulated with anti-CD3 and anti-CD28 were cocultured with LSECs cell contact was required for suppression. We then tested whether from C57BL/6 mice. LSECs pretreated with poly(I:C), LPS, or LSECssuppressedTcellsbyactivatingprogrammeddeath1(PD-1), IFN-g behaved like untreated LSECs and significantly inhibited an inhibitory receptor expressed on T cells. The suppression of T cell T cell proliferation and cytokine production of anti-CD3/anti- activation by LSECs was abolished by pretreating LSECs with a CD28–activated cells (Fig. 1A). In contrast, pretreatment of LSECs neutralizing Ab to the ligand of PD-1, PD-L1 (Supplemental Fig. 1D), with P3C not only abrogated the suppression of T cell activation which is constitutively expressed on the surface of LSECs. by LSECs, but even enhanced T cell proliferation and cytokine production compared with T cell cultures without LSECs (Fig. Pretreatment of LSECs with P3C, but not poly(I:C) or LPS, 1A). The enhanced IFN-g production by activated T cells, after reverts their suppressive properties and allows T cell activation cocultivation with P3C-stimulated LSECs, was dependent on the TLR ligands can induce phenotypic and functional maturation of concentrations of P3C applied to LSECs. A low dose of 0.1 mg/ml APCs, characterized by an increased expression of costimulatory P3C was already sufficient to abrogate the LSEC-mediated sup- molecules, and promote immunity (4). Therefore, we investigated pression of T cell functions (Fig. 1B). Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. TLR1/2 ligand (P3C)-stimulated LSECs further enhance the activation of T cells. (A) LSECs were stimulated with 10 mg/ml P3C (TLR1/2), 10 mg/ml poly(I:C) (TLR3), 10 mg/ml LPS (TLR4), or 50 ng/ml IFN-g for 24 h. Polyclonal stimulated splenocytes were cocultured with pretreated LSECs at a ratio of 1:2 (LSECs:splenocytes), and T cell cytokine production and proliferation were measured after 48 h. Anti-CD3/anti-CD28–stimulated splenocytes only were used as responder controls. Unstimulated splenocytes were used as negative control (NC). (B) LSECs were stimulated with different concentrations of P3C (1 ng/ml–100 mg/ml) for 24 h. Polyclonal stimulated splenocytes were cocultured with pretreated LSECs. T cell cytokine production was measured after 48 h. Data shown are the mean 6 SD of one of three representative experiments. *p , 0.05, **p , 0.01, ***p , 0.001. 4 ACTIVATION OF LSECs BY TLR1/2 LIGANDS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Transition of tolerogenic to immunogenic LSECs after TLR1/2 stimulation. LSECs were treated with TLR ligands, IFN-g, or left untreated for 24 h, washed, and pulsed with 2 mg/ml FV peptide (FV GagL CTL epitope aa 85–93). CFSE-labeled naive FV-TCR TCR tg CD8+ T cells were cocultured with those LSECs or cultured alone [green lines in (A)]. As positive control, the T cells were cocultured with bone marrow–derived FV peptide-pulsed DCs. After 72 h, analysis for T cell proliferation (A) and for effector T cell markers (CD25, CD44, CD62L, CD69, and granzyme B) (B) was performed by flow cytometry. The numbers in (A) indicate the proliferation index. Representative histograms are shown. (C) The production of the cytokines IFN-g, IL-2, and TNF-a by the FV-TCR CD8+ T cells was measured by ELISA after 72 h of coculture (upper panel). The IFN-g production by T cells was also measured by intracellular cytokine staining after 7 d of coculture (lower panel). (D) Activated FV-TCR TCR tg CD8+ T cells were analyzed for their cytotoxic potential against FV peptide-loaded EL4 cells in an in vitro kill assay. (E) LSECs were pulsed with 2 mg/ml WHcAg peptide (WHcAg CTL epitope aa 13–21) and were cocultured with WHcAg-specific CD8+ T cells purified from WHcAg-immunized mice. IFN-g production by T cells was measured either by ELISA after 3 d of coculture (upper panel) or by intracellular cytokine staining after 7 d of coculture (lower panel). The numbers (C, D) indicate the percentage of positive cells; APCs/effectors = 1:2. Data shown are the mean 6 SD of one of three representative experiments. *p , 0.05, **p , 0.01. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. LSEC-triggered CD8+ T cell immunity is mediated by IL-12. (A) Upper panel, LSECs were pretreated with 10 mg/ml P3C for 24 h, washed, and separately cocultured with polyclonal stimulated splenocytes in a Transwell plate system. Lower panel, LSECs were incubated with 10 mg/ml P3C for 24 h, washed, and cultured for another 24 h. Supernatant of the LSECs was then harvested and transferred to the polyclonal stimulated splenocytes that were cocultured with untreated LSECs (TLR1/2-ST). Cytokine production by T cells was measured by ELISA after 48 h. (B) Polyclonal stimulated splenocytes were cocultured with TLR1/2-stimulated LSECs, and 5 mg/ml anti–IL-12 Abs, anti–IL-23 Abs, or isotype control Abs were added to the cocultures. Cytokine production by T cells was measured by ELISA after 48 h. (C) Polyclonally stimulated splenocytes were cocultured with TLR1/2- stimulated LSECs, and 5 mg/ml anti–IL-12 Abs (anti–IL-12), anti–IL-23 Abs (anti–IL-23), or isotype control Abs (Control Ab) were added to the cocultures. T cell proliferation was measured after 48 h. (D) Polyclonal stimulated splenocytes were cocultured with TLR1/2-stimulated LSECs, and different concentrations of rIL-12 as indicated were added to the cocultures. (E) Naive FV-TCR TCR tg CD8+ T cells were (Figure legend continues) 6 ACTIVATION OF LSECs BY TLR1/2 LIGANDS

FIGURE 4. Induction of IL-12 production by LSECs after TLR ligand stimulation. (A) LSECs were stimulated by TLR ligands or IFN-g for either 6 or 24 h. IL-12 expression by LSECs was deter- mined either at the RNA level by real-time RT-PCR (6-h stimulation) or at the protein level (24-h stimulation) by ELISA. (B) LSECs were stimulated by 10 mg/ml P3C (TLR1/2) or 10 mg/ml LPS (TLR4) for 24 h (day 0), washed, and cultured for another 8 d without stimulation. Culture medium was changed every 24 h. IL-12 secretion by LSECs was determined by ELISA. Results are representa- tive of two independent experiments. Downloaded from http://www.jimmunol.org/

Induction of Ag-specific CD8+ T cell immunity by LSECs after The ability of TLR-stimulated LSECs to restimulate vaccine- TLR1/2 ligand stimulation primed CD8+ T cells was also analyzed. The CD8+ T cells were The previous results indicated that LSECs are able to activate T cells primed in mice by three immunizations with a DNA vaccine and promote T cell functions if LSECs were stimulated by TLR1/2 expressing WHcAg. Splenocytes were isolated from these mice ligand. Thus, TLR1/2-stimulated LSECs may present Ag to naive and cocultured with TLR-stimulated LSECs loaded with the T cells and elicit Ag-specific CD8+ T cell immunity. To verify this WHcAg immundominant CTL epitope peptide YQLLNFLPL. + Again, only P3C-stimulated LSECs, but not poly(I:C)-, LPS-, or hypothesis, we explored TCR tg CD8 Tcellsspecifictothe + DbGagL FV epitope in the mouse model (FV-TCR CD8+ T cells). IFN-g–treated LSECs, could restimulate WHcAg-specific CD8 by guest on October 2, 2021 FV infection model represents a well-established animal model to T cells in vitro to produce large amounts of IFN-g (Fig. 2E). + Taken together, these data show that LSECs are able to effi- study T cell immunity to retrovirus (18). FV-TCR CD8 T cells + were cocultured with untreated versus treated epitope peptide- ciently present Ag and activate Ag-specific CD8 T cells after loaded LSECs. In contrast to poly(I:C), LPS, or IFN-g, P3C was TLR1/2 stimulation. able to stimulate LSECs that in turn induced a significantly in- creased proliferation of FV-TCR CD8+ T cells, comparable to the TLR1/2 signaling does not increase the expression of proliferation of tg T cells activated by bone marrow–derived den- costimulatory molecules or PD-L1 on LSECs dritic cells (bmDCs) (Fig. 2A). The FV-TCR CD8+ Tcellsprimed To clarify the mechanisms of the functional maturation of LSECs by by P3C-stimulated LSECs showed an activated effector phenotype TLR1/2 stimulation, the expression levels of costimulatory and in- similar to T cells primed by immunogenic bmDCs. These activated hibitory molecules CD80, CD86, and PD-L1 on LSECs were ex- T cells showed an increased expression of CD25, CD44, CD69, and amined. Neither TLR nor IFN-g stimulation could upregulate the granzyme B, and decreased CD62L compared with T cells cocul- expression of costimulatory molecules CD80 and CD86. On the tured with untreated, peptide-loaded LSECs (Fig. 2B). contrary, P3C, poly(I:C), and LPS stimulation slightly downregulated The lack of IFN-g expression after TCR stimulation is char- CD80 and CD86 expression at both protein and RNA levels in acteristic for T cells tolerized by LSECs (13). To test the capacity LSECs. However, unlike LPS and IFN-g stimulation, P3C and of TLR-stimulated LSECs to induce T cell immunity, LSECs poly(I:C) stimulation did not upregulate PD-L1 expression on LSECs loaded with FV peptide were first stimulated with TLR ligands or (Supplemental Fig. 2A, 2B). In vivo TLR1/2 stimulation was also IFN-g and then cocultured with FV-TCR CD8+ T cells. Only P3C- performed by i.p. injecting 100 mg P3C into mice. LSECs and splenic stimulated LSECs promoted a differentiation of naive FV-TCR DCs were purified from those mice 24 h after injection and stained CD8+ T cells into IFN-g–producing effector cells as measured for CD80, CD86, and PD-L1. No upregulation of CD80, CD86, and by ELISA and intracellular cytokine staining (Fig. 2C). These PD-L1 expression on the LSECs in response to in vivo TLR1/2 stimu- CD8+ T cells also produced IL-2 and TNF-a (Fig. 2C) and showed lation was observed either (Supplemental Fig. 2C). In contrast, the cytotoxic activity as they efficiently killed epitope peptide-loaded CD80 and CD86 expression on splenic DCs was significantly up- target cells in vitro (Fig. 2D). regulated after in vivo P3C treatment (Supplemental Fig. 2D).

cocultured with 2 mg/ml FV peptide-pulsed LSECs in the presence of different concentrations of IL-12, as indicated. Subsequently, IFN-g production by T cells was measured either by ELISA after 3 d of culture (left panel) or by intracellular cytokine staining after 7 d of culture (right panel). Data shown are the mean 6 SD of one of three representative experiments. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7

TLR1/2-stimulated LSECs do not inhibit activation-induced through FV-TCR using annexin V and 7-aminoactinomycin D CD8 T cell apoptosis staining. In the anti-CD3/anti-CD28–mediated T cell activation, no Activation-induced T cell apoptosis is believed to limit T cell difference of early apoptosis of CD8 T cells was observed when proliferation and eliminate the high number of activated T cells T cells were cultured alone, cocultured with unstimulated LSECs, during host immune responses. Next, we examined whether TLR1/ or cocultured with P3C-stimulated LSECs. There was increased 2-stimulated LSECs had influence on the apoptosis of CD8 T cells end-stage apoptosis and CD8 T cell death when T cells were during their activation. Apoptosis of T cells was measured in acti- cocultured with P3C-stimulated LSECs (Supplemental Fig. 3A). vation by anti-CD3/anti-CD28 treatment and Ag-specific activation After Ag-specific activation of FV-TCR CD8 T cells, CD8 T cells Downloaded from http://www.jimmunol.org/

FIGURE 5. Unique transcriptional signature of P3C- stimulated LSECs. (A) Part of the differently regulated genes between P3C-stimulated LSECs and poly(I:C), LPS-, or IFN-g–stimulated LSECs are shown. The regulation degrees of genes are indicated by differ- by guest on October 2, 2021 ent colors. Significantly regulated genes were selected using the following criteria: fold change .2 (log2 ratio $ 61), FDR ,0.001. (B) Statistic chart of differ- entially expressed genes (DEGs) in LSECs after the stimulation with TLR ligands or IFN-g. The numbers of genes that were significantly regulated in P3C-, poly(I:C)-, LPS-, or IFN-g–stimulated LSECs (com- pared with untreated LSEC) are shown. Significantly regulated genes were selected using the following cri- teria: fold change .2 (log2 ratio $ 61), FDR ,0.001. 8 ACTIVATION OF LSECs BY TLR1/2 LIGANDS primed by LSECs showed decreased early apoptosis compared and only soluble factors could pass through the membrane. P3C- with T cells primed by DCs. P3C treatment on LSECs had no stimulated LSECs maintained their ability to stimulate the cyto- influence on early apoptosis, but slightly decreased end-stage kine production by anti-CD3/anti-CD28–activated T cells in the apoptosis and death of CD8 T cells (Supplemental Fig. 3B). Transwell (Fig. 3A). Transfer of the supernatants of P3C-stimulated This result indicated that CD8 T cell activation by TLR1/2- LSECs into cocultures of activated T cells and untreated LSECs stimulated LSECs was not correlated with the regulation of significantly increased the cytokine production by activated T cells T cell apoptosis. (Fig. 3A). These data clearly indicated that P3C-stimulated LSECs

+ triggered T cell immunity by producing soluble factors. Because LSEC-induced CD8 T cell immunity is partially mediated by IL-12 is known to be a potent soluble costimulatory signal and is IL-12 production required to promote cytotoxic CD8+ T cell differentiation, we sus- As the costimulatory molecules were not upregulated on P3C- pected that P3C stimulated LSECs to produce IL-12 and thereby stimulated LSECs, we assumed that the observed immunostimu- induced T cell activation. Therefore, an IL-12–blocking Ab was lation for T cells may be mediated by soluble factors. Conse- used to neutralize IL-12 in the coculture system. When IL-12 was quently, P3C-stimulated LSECs were cocultured with anti-CD3/ blocked, P3C-stimulated LSECs completely lost their potential anti-CD28–activated T cells in a Transwell system. In this system, to promote IFN-g production by activated T cells, but suppressed LSECs and T cells were separated by a semipermeable membrane, the cytokine production (Fig. 3B). IL-12 was secreted by the Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. TLR1/2 stimulation reverts suppressive properties of LSECs with PD-L1high CD80/86low phenotype upon T cell activation. (A) LSECs were stimulated by 50 ng/ml IFN-g for 24 h, washed, and pulsed with 2 mg/ml FV peptide. The pretreated LSECs were then cocultured with CFSE-labeled naive FV- TCR CD8+ T cells in the presence of different concentrations of IL-12, as indicated. Flow cytometric analysis was performed to measure the proliferation of CFSE-labeled CD8+ T cells after 72 h. Green lines indicate the proliferation of CD8+ T cells that were cultured alone. The numbers indicate the proliferation index. (B) LSECs in (A) were either left untreated or treated with 10 mg/ml PD-L1 Ab for 1 h before coculture with CD8+ T cells. Cytokine production by T cells was measured by ELISA after 3 d of coculturing. (C) LSECs were first stimulated with 50 ng/ml IFN-g for 24 h, stimulated with 10 mg/ml P3C for another 24 h, and then washed. The pretreated LCESs were cultured with polyclonal stimulated splenocytes. The cytokine production and proliferationof splenocytes were measured after 48 h. Data shown are the mean 6 SD of one of three representative experiments. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 9 stimulated LSECs and not by the T cells themselves, as IL-12– rogated in the presence of rIL-12. This was shown by measuring blocking Ab showed no effect on IFN-g production in cultures IFN-g in the supernatants and by intracellular cytokine staining in containing only activated T cells without LSECs. Because the FV-TCR CD8+ T cells (Fig. 3E). In summary, these experiments IL-12–blocking Ab used can also neutralize IL-23, a related cy- indicate that TLR1/2-stimulated LSECs augmented CD8+ T cell tokine with the ability to modulate T cell functions, we addressed immunity through the secretion of soluble factors, such as IL-12 the role of this cytokine in our cultures. An IL-23–specific Ab was and likely other cytokines. added to neutralize secreted IL-23. In contrast to the IL-12 block- ade, IL-23 neutralization showed no effect on IFN-g production by Weak IL-12 production by LSECs after TLR stimulation activated T cells (Fig. 3B). IL-12 blockade also abolished the fur- IL-12 production by TLR-stimulated LSECs was further ex- ther enhancement of T cell proliferation by P3C-stimulated LSECs. amined at both the RNA and protein levels. Elevated IL-12 In the presence of IL-12–blocking Ab, the proliferation of T cells mRNA levels and IL-12 proteins in the supernatant of LSECs cocultured with P3C-stimulated LSECs decreased to a comparable after TLR1/2 or TLR4 stimulation were detected (Fig. 4A). A level to the activated T cells cultured along (Fig. 3C). This result high level of IL-12 production was stimulated by TLR4 stim- indicates that, in addition to IL-12, other cytokines may also be ulation within the first 24 h. However, LSECs quickly lost their involved in the P3C-stimulated LSEC-augmented T cell prolifer- ability to produce IL-12 without sustained TLR4 stimulation. ation. In contrast, adding rIL-12 to cocultures of anti-CD3/anti- On the contrary, TLR1/2-stimulated LSECs continuously se- CD28–activated T cells and unstimulated LSECs abrogated the creted IL-12 for 1 wk after initial stimulation for 24 h (Fig. 4B). suppression of T cell IFN-g production by LSECs. Interestingly, Notably, although TLR3- and TLR1/2-stimulated LSECs show even a low concentration of IL-12 at 1 pg/ml was sufficient to over- a similar pattern of the expression of costimulatory molecules come the suppressive effect of LSECs on IFN-g production by (Supplemental Fig. 2C), TLR3-stimulated LSECs totally lack Downloaded from T cells (Fig. 3D). To verify these results with Ag-specific T cells, IL-12 production in comparison with TLR1/2-stimulated LSECs. naive FV-TCR TCR tg CD8+ T cells were cocultured with unsti- This lack of IL-12 production could be the major reason that TLR3- mulated FV peptide-loaded LSECs. Again, the LSEC-mediated stimulated LSECs are unable to activate T cells. These results dem- suppression of IFN-g production by Ag-specific T cells was ab- onstrated that P3C stimulation activated LSECs in a unique way. http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 7. Synergistic effect of TLR1/2 stimulation and PD-L1 blockade on triggering CD8+ T cell immunity by LSECs. LSECs were first either left unstimulated or stimulated with 10 mg/ml P3C for 24 h, and then either left untreated or treated with 10 mg/ml PD-L1 Ab for 1 h. Cells were pulsed with 2 mg/ml FV peptide, washed, and cocultured with naive FV-TCR TCR tg CD8+ T cells. The cytokine production by T cells was measured by ELISA after 72 h. Data shown are the mean 6 SD of one of two representative experiments. *p , 0.05, **p , 0.01, ***p , 0.001. 10 ACTIVATION OF LSECs BY TLR1/2 LIGANDS

Distinct transcriptional signatures of LSECs after stimulation function of primed FV-TCR CD8+ T cells could be tested directly. with different TLR ligands Naive FV-TCR CD8+ T cells were primed with FV epitope peptide- To further characterize the transition from a tolerogenic to an im- loaded LSECs that were either untreated or P3C stimulated in vitro. munogenic APC after treatment with TLR1/2 ligand, we compared FV peptide-loaded bmDCs served as a positive control. The stim- the transcriptional signature of differently stimulated LSECs. High ulated T cells were then harvested, washed, and transferred into congenic mice acutely infected with FV. The number of trans- throughput sequencing was performed to define the transcriptomes of + the LSECs. Cross-annotation of the transcription signatures of P3C- ferred FV-TCR CD8 T cells preprimed by P3C-treated LSECs stimulated LSECs and unstimulated LSECs yielded 619 differen- and bmDC in spleens of recipient mice increased significantly, tially expressed transcripts, coding for 547 annotated murine genes indicating a substantial expansion of the transferred T cells (Fig. 8A). Consistently, a significant reduction in viral loads in spleens (Supplemental Table I). Compared with poly(I:C), LPS, or IFN-g,P3C + stimulation of LSECs induced a unique transcription signature of of recipient mice occurred in mice that received FV-TCR CD8 T cells preprimed by P3C-pretreated LSECs and bmDC (Fig. 8B). the cells (Fig. 5A); one notable feature of it is that significantly less + genes were downregulated (Fig. 5B). Because P3C-stimulated LSECs FV-TCR CD8 T cells preincubated with untreated LSECs did triggered T cell immunity by producing soluble factors, the genes not exhibit an expansion and any anti-FV activity in vivo. These related to cytokine–cytokine receptor interaction were analyzed. The results clearly demonstrated that TLR1/2 stimulation of LSECs results suggested that P3C-stimulated LSECs have distinct features can shift T cell tolerance to antiviral T cell immunity (Fig. 9). of cytokine production and cytokine receptor regulation compared with poly(I:C), LPS, or IFN-g–stimulated LSECs (data not shown), + which may be important for the activation of CD8 T cells. Downloaded from TLR1/2 stimulation fully reverts suppressive properties of LSECs with PD-L1high CD80/86low phenotype It has been shown that PD-L1 expression in the liver is upregulated during chronic hepatitis viral infection or hepatocellular carcinoma (25, 26). Thus, it is important to analyze whether TLR1/2 stim- ulation or additional IL-12 can also abrogate the suppression of http://www.jimmunol.org/ T cell activation by LSECs with high levels of PD-L1 expression. As LSECs strongly upregulate PD-L1 expression in response to IFN-g stimulation, LSECs were stimulated with IFN-g to gener- ate LSECs with PD-L1high CD80/86low phenotype. The PD-L1high LSECs were cocultured with FV-TCR CD8+ T cells in the pres- ence of the FV peptide at a concentration of 2 mg/ml. Different concentrations of IL-12 were added during the cocultivation. Sup-

plementation of IL-12 potently enhanced IFN-g production by by guest on October 2, 2021 FV-TCR CD8+ T cells cocultured with PD-L1high LSECs (Fig. 6B). However, IL-12, even at a concentration of 1 ng/ml, could not abrogate the suppression of T cell proliferation (Fig. 6A) and IL-2 and TNF-a production by IFN-g–stimulated LSECs (Fig. 6B). PD-L1 blockade by anti–PD-L1 was required to restore T cell IL-2 and TNF-a production (Fig. 6B). These results demonstrated that IL-12 stimulation alone was not enough to overcome the sup- pressive effects mediated by PD-L1high LSECs. In contrast, when PD-L1high LSECs were stimulated with P3C for 24 h before co- culture with anti-CD3/anti-CD28–activated T cells, they were able to enhance IFN-g, IL-2, and TNF-a production by activated FV- TCR T cells, as well as T cell proliferation (Fig. 6C). Synergistic effects of TLR1/2 stimulation and PD-L1 blockade on triggering CD8+ T cell immunity by LSECs Based on the previous experiments, we assumed that a synergistic effect on triggering T cell immunity may be achieved by combining TLR1/2 stimulation of LSECs with PD-L1 blockade. LSECs were FIGURE 8. TLR1/2-stimulated LSEC-primed FV-TCR CD8+ T cells are pretreated with P3C and/or anti–PD-L1, loaded with FV peptide, able to proliferate and reduce acute FV infection in vivo. FV-TCR TCR tg and cocultured with FV-TCR CD8+ T cells. Combining P3C and CD8+ T cells (CD45.1+) were cocultured with 2 mg/ml FV peptide-pulsed anti–PD-L1 significantly increased FV-TCR CD8+ T cell prolif- LSECs that were either treated with 10 mg/ml P3C (TLR1/2-LSEC) or left eration as well as IFN-g, IL-2, and TNF-a production, compared untreated (unsti-LSEC). As positive control, the T cells were cocultured with with controls with only P3C stimulation or Abs to PD-L1 (Fig. 7). bone marrow–derived 2 mg/ml FV peptide-pulsed DCs (DC). After 72 h, FV- TCR TCR tg CD8+ T cells were harvested and washed with PBS, and 5 3 Antiviral functions of FV-TCR CD8+ T cells primed by TLR1/ 106 purified T cells were adoptively transferred into recipient mice (CD45.2), 2-stimulated LSECs in vivo which were at day 5 post-FV infection. (A) The numbers of CD45.1-positive cells per 106 splenocytes in the recipient mice were counted by flow The crucial question is whether TLR1/2-stimulated LSECs indeed cytometry at 3 d after adoptive transfer. (B) Viral loads in the spleen of + prime Ag-specific CD8 T cells with antiviral functions in vivo. To infected mice were calculated by infectious center assay at 3 d after adoptive answer this question, we further explored the FV infection mouse transfer of the T cells. Data shown are the mean 6 SD of one of two rep- model to test the T cell functions. In the FV infection system, the resentative experiments. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 11

Discussion IL-12 is well documented to serve as so-called signal 3 cytokine, In this study, we investigated how TLR1/2 ligand P3C influences capable of facilitating CD8+ T cell proliferation, effector func- the functions of LSECs and demonstrated that LSECs display tions, and memory formation (28). Therefore, it was not surprising functional plasticity with respect to induction of tolerance and that IL-12 secretion by LSECs could induce T cell immunity. immunity. To maximally mimic the physiological conditions in the However, other unidentified soluble mediator(s) may also partic- liver, we used primary LSECs isolated by classic isolation and ipate in TLR1/2 ligand-mediated T cell activation by LSECs. Both cultivation method that can highly maintain the typical in situ TLR1/2 ligand and TLR4 ligand stimulation can induce IL-12 characteristics of the cells (27). Besides, diverse T cell activation production by LSECs, but TLR1/2-stimulated LSECs exhibit to- systems were used to mimic different activation statues T cells tally different kinetics of IL-12 production after the ligand was may have during a classic immune response, including polyclonal removed. TLR4-stimulated LSECs quickly lost their ability to TCR-activated T cells (anti-CD3/anti-CD28 stimulation) and Ag- produce IL-12 in the absence of stimulation. On the contrary, specific TCR-activated T cells (FV-TCR tg CD8+ T cells), as well TLR1/2-stimulated LSECs produced IL-12 for at least 1 wk. Dif- as naive T cells and primed T cells (WHcAg-experienced T cells). ferent IL-12 production patterns between these two TLR stim- In these in vitro coculturing systems, LSECs potently suppress the ulations may explain why only TLR1/2-stimulated LSECs are able proliferation and cytokine production by TCR-activated T cells to promote the activation of T cells, as it has previously been via PD-1/PD-L1 interaction. However, this immunosuppressive shown that full activation of CD8+ T cells requires IL-12 signaling property is abrogated by TLR1/2, but not by TLR3 or TLR4 stim- for .40 h (29). ulation. More important, TLR1/2-stimulated LSECs promoted It is likely that the balance of the signals delivered through differentiation of naive into functional effector CD8+ T cells with costimulatory and inhibitory molecules on T cells determines in- Downloaded from the ability to produce cytokines, kill specific target cells, and sup- duction of immunity or tolerance (11). Therefore, a combination of press FV replication in vivo. However, TLR1/2 signaling did not costimulatory stimulation and inhibitory blockage should achieve upregulate costimulatory molecules, like CD80/CD86 or CD40 a synergistic effect on T cell activation, which has been shown in (data not shown), on LSECs, similar to the results from previous chronic viral infection and tumor therapy (30, 31). In the current studies (14). Notably, in contrast to TLR4 and IFN-g stimulation, study, we demonstrate that the combination of TLR1/2 stimulation TLR1/2 stimulation did not increase PD-L1 expression on LSECs. and PD-L1 blockage has a synergistic effect on LSEC-induced This fact may partially facilitate T cell activation. The activating CD8 T cell activation. Such a new strategy may overcome im- http://www.jimmunol.org/ effect of the TLR1/2 ligand was mediated by soluble components munological tolerance in situations like persistent viral infection with IL-12 as one major mediator. of the liver or liver cancer. by guest on October 2, 2021

FIGURE 9. Mechanism of regulation of T cell activation by LSECs. (A) LSECs strongly upregulate PD-L1 expression in response to the IFN-g produced by activated T cells and subsequently induce T cell tolerization. (B) The presence of specific pathogens induces the functional maturation of LSECs, for example, producing IL-12 by TLR1/2-stimulated LSECs, which allow T cells to mount an immune response. 12 ACTIVATION OF LSECs BY TLR1/2 LIGANDS

One concern that has to be mentioned in this study is that most (Fig. 9). The special role of LSECs in regulating T cell activation experiments were carried out by using LSECs cultured in vitro. in the liver makes them attractive therapeutic targets. Strategies LSECs may change their characteristics when grown under in vitro that promote LSEC-mediated immunosuppression could reduce culture conditions. For example, the presence of transcytoplasmic autoimmune disease and enhance transplantation tolerance. Alter- fenestrations is one of the hallmark features used to identify LSECs natively, therapies that reduce the suppressive effects of LSECs, and defines them as a specialized liver-specific endothelial cell. such as treatment with P3C in combination with PD-L1 blockage, However, in vitro cultured LSECs typically undergo defenestration could boost immune responses and may lead to more effective quickly (32). It has also been shown previously that, when cul- treatment against liver infection and cancer. In summary, the ability tured alone after isolation, rat LSECs lose the expression of SE-1, of LSECs to contribute to both immunity and tolerance highlights increase expression of CD31, and undergo apoptosis after 2 d (33, their considerable therapeutic potential, as well as the importance 34). How these changes of cultured LSECs influence the outcomes of increasing our understanding of how APCs function in the liver of their immunological functions is to be determined yet. To ad- environment. dress the real situation of LSECs in response to TLR stimulation in vivo, further studies need to be performed by using sophisti- Acknowledgments cated tg mouse models with LSECs as the main cell population We thank Daniela Catrini and Dr. Anna Kosinska for critical reading of this capable of cross-presenting certain peptide to Ag-specific CD8 manuscript. T cells. Another concern of in vivo P3C treatment is the function of which other APCs in the liver may be influenced. In our previous Disclosures studies (7, 35, 36), liver cells such as Kupffer cells, stellate cells, The authors have no financial conflicts of interest. Downloaded from and hepatocytes were found to functionally express TLR1/2. Kupffer cells and splenic DCs showed similar abilities to enhance CD8 References T cell activation in response to TLR1/2 stimulation. TLR1/2 stim- 1. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. ulation also induces the production of proinflammatory cytokines 4: 499–511. and mediates innate immune responses in hepatocytes. Therefore, 2. Reis e Sousa, C. 2004. Toll-like receptors and dendritic cells: for whom the bug Semin. Immunol. tolls. 16: 27–34. http://www.jimmunol.org/ LSECs mediate the TLR1/2 stimulation along with other hepatic 3. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of cell types. Very recent study has demonstrated signaling via TLRs immunity. Nature 392: 245–252. induced intrahepatic aggregates of myeloid cells that enabled the 4. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197–216. population expansion of CTLs (37). It has also been shown that 5. Trinchieri, G. 2003. Interleukin-12 and the regulation of innate resistance and liver-primed T cells are reactivated from their nonresponsive state adaptive immunity. Nat. Rev. Immunol. 3: 133–146. 6. Thompson, A. J., D. Colledge, S. Rodgers, R. Wilson, P. Revill, P. Desmond, and develop into effector CTLs upon TCR stimulation in combi- A. Mansell, K. Visvanathan, and S. Locarnini. 2009. Stimulation of the nation with costimulatory signals via CD28 and IL-12 (38). Thus, interleukin-1 receptor and Toll-like receptor 2 inhibits hepatitis B virus repli- the contribution of different liver cells, especially APCs that possess cation in hepatoma cell lines in vitro. Antivir. Ther. 14: 797–808. 7. Wu, J., Z. Meng, M. Jiang, E. Zhang, M. Trippler, R. Broering, A. Bucchi, the ability to produce IL12 (such as Kupffer cells and DCs), to

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Supplemental figure 1. LSECs suppress T cell cytokine production and proliferation via PD-1/PD-L1 interaction. (A) LSECs were stained by either isotype control antibodies or anti-CD146, anti-NK1.1, anti-DX5, anti-CD11b, anti-CD11c, and anti-F4/80 after magnetic microbead purification. The purity of the cell fractions was then measured by flow cytometry. (B) Unstimulated or anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) stimulated splenocytes from C57BL/6 mice were cultured with or without LSECs at a ratio of 1:2 (LSECs:splenocytes). T cell cytokine production and proliferation were measured after 48 h. Anti-CD3/CD28 stimulated splenocytes only were used as responder controls (RC). Unstimulated splenocytes alone were used as negative control (NC). Unstimulated splenocytes cocultured with LSECs were used as unstimulated control (UC). (C) LSECs were cocultured with polyclonal-stimulated splenocytes in contact or transwell (LSECs and splenocytes separated by a semipermeable membrane) 96-well plates. T cell cytokine production and proliferation were measured after 48 h. (D) LSECs were incubated with 10 μg/ml isotype control antibodies (iso Ab) or anti-PD-L1 antibody (αPD-L1) for 1 h. Polyclonal stimulated splenocytes were cultured with antibodies treated LSECs. T cell cytokine production and proliferation were measured after 48 h. Data shown are the mean ± SD of one out of 3 representative experiments. *p < 0.05; **p < 0.01.

Supplemental figure 2. TLR1/2 signaling does neither increase the costimulatory molecules expression nor the PD-L1 expression on LSECs. LSECs were stimulated in vitro by 10 μg/ml P3C(TLR1/2), 10 μg/ml poly(I:C)(TLR3), 10 μg/ml LPS(TLR4), or 50 ng/ml IFN-γ for either 24 h (A) or 6 h (B). (A) The changes of CD80, CD86, and PD-L1

1

expression on cells were then determined by flow cytometry. (B) The changes of CD80,

CD86, and PD-L1 expression at the RNA level were determined by real-time RT-PCR.

Results are representative of two independent experiments. (C) C57BL/6 mice were intraperitoneally injected with PBS or 100 μg P3C. LSECs from those animals were obtained 24 h later. The expression of CD80, CD86, and PD-L1 was determined by flow cytometry. (D) C57BL/6 mice were intraperitoneally injected with PBS or 100 μg P3C.

Splenic DCs from those animals were obtained 24 h later. The expression of CD80 and

CD86 was determined by flow cytometry.

Supplemental figure 3. TLR1/2 stimulated LSECs do not inhibit activation induced

CD8 T cell apoptosis. (A) Unstimulated or anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) stimulated splenocytes from C57BL/6 mice were cultured with or without LSECs at a ratio of 1:2 (LSECs:splenocytes). CD 8 T cell apoptosis and death were analyzed by using Annexin V and 7-AAD staining after 48 h. Anti-CD3/CD28 stimulated splenocytes only were used as responder controls (RC). Unstimulated splenocytes alone were used as negative control (NC). (B) P3C pretreated or untreated LSECs pulsed with 2 µg/ml FV peptide (FV GagL CTL epitope aa 85-93). Naïve FV-TCR TCR transgenic CD8+ T cells were cocultured with those LSECs or cultured alone (NC). As positive control (PC), the T cells were cocultured with bone marrow-derived FV peptide-pulsed DCs. CD 8 T cell apoptosis and death were analyzed by using Annexin V and 7-AAD staining after 72 h.

Annexin V positive and 7-AAD negative cells are considered as early apoptosis cells and

Annexin V and 7-AAD double positive cells are considered as end stage apoptosis and death cells.

2

Supplemental table 1. Gene expression difference analysis in P3C stimulated LSECs. In

the table, the fold changes (log2 Ratio) of genes, which were significantly regulated in

P3C stimulated LSECs (compared to untreated LSEC), are shown. Significantly regulated

genes were selected using the following criteria: fold change>2 (Log2 Ratio≥ ±1), false discovery rate (FDR)<0.001.

3 A

n.s. n.s. B * 60000 ** 150000 C 60000 150000 * ** 40000 100000 40000 100000 pg/ml pg/ml   20000 50000 20000 50000 IFN- IFN- Proliferation cpm Proliferation cpm 0 0 0 0 NC UC RC LSEC NC UC RC LSEC RC Contact Transwell RC Contact Transwell ¢CD3/¢CD28 - - + + ¢CD3/¢CD28 - - + + LSEC - + - + LSEC - + - + n.s. n.s. 5000 * 800 * * 5000 * 800 4000 4000 600 600 3000 3000 pg/ml pg/ml

 400

 400 2000 2000 IL-2 pg/ml IL-2 pg/ml TNF- 200 TNF- 200 1000 1000

0 0 0 0 NC UC RC LSEC NC UC RC LSEC RC Contact Transwell RC Contact Transwell ¢CD3/¢CD28 - - + + ¢CD3/¢CD28 - - + + LSEC - + - + LSEC - ++-

n.s. n.s. D 50000 150000 ** ** 40000 100000 30000 pg/ml 20000  50000 IFN- 10000 Proliferation cpm Proliferation 0 0 RC Iso Ab PD-L1 RC Iso Ab PD-L1

n.s. n.s. ** 8000 1000 * 800 6000 600 pg/ml

4000  400 IL-2 pg/ml IL-2 2000 TNF- 200

0 0 RC Iso Ab PD-L1 RC Iso Ab PD-L1

Supplementary Table 1: Gene expression difference analysis in P3C stimulated LSECs. In the table, the fold changes (log2 Ratio) of genes,which were significantly regulated in P3C stimulated LSECs (compared to untreated LSEC) are shown. Significantly regulated genes were selected using the following criteria: fold change>2 (Log2 Ratioı ±1), false discovery rate (FDR)<0.001.

Symbols Gene names log2 Ratio Acpp acid phosphatase, prostate, isoform CRA_b 9.44 Gm6578 39S ribosomal protein L32, mitochondrial 9.40 Il1b interleukin-1 beta precursor 6.61 Pls1 plastin-1 5.86 Depdc1a DEP domain-containing protein 1A isoform 3 5.16 Taz tafazzin, isoform CRA_c 4.52 Chgb secretogranin-1 precursor 4.20 Nit1 nitrilase homolog 1 isoform 1 4.17 Ccl20 C-C motif chemokine 20 isoform 1 4.02 Fpr1 fMet-Leu-Phe receptor 3.81 Mmp13 collagenase 3 preproprotein 3.57 Rgs4 regulator of G-protein signaling 4 3.46 Il1a interleukin 1 alpha, isoform CRA_a 3.21 Nox4 NADPH oxidase 4 3.02 Ccl3 C-C motif chemokine 3 2.88 Kcnh1 potassium voltage-gated channel subfamily H member 1 isoform 2 2.83 Clec4a1 C-type lectin domain family 4, member a1 2.81 Cxcl5 C-X-C motif chemokine 5 precursor 2.78 Steap4 metalloreductase STEAP4 2.76 Il18r1 interleukin-18 receptor 1 isoform a 2.74 Cenpe centromere-associated protein E 2.69 Fpr2 formyl peptide receptor 2 2.69 Serf1 small EDRK-rich factor 1 2.64 Gm6377 novel protein 2.58 Cenph centromere protein H 2.58 Irg1 immune-responsive gene 1 2.58 Kif18a kinesin-like protein KIF18A 2.58 Cnr1 cannabinoid receptor 1 2.56 9930023K05Rik RIKEN cDNA 9930023K05, isoform CRA_c 2.56 Pilra paired immunoglobulin-like type 2 receptor alpha precursor 2.55 Nusap1 nucleolar and spindle-associated protein 1 isoform a 2.53 4930547N16Rik mCG16776, isoform CRA_b 2.52 Esco2 N-acetyltransferase ESCO2 2.48 Hjurp Holliday junction recognition protein 2.44 Ccl9 C-C motif chemokine 9 2.40 Cxcl2 C-X-C motif chemokine 2 precursor 2.38 Fam64a family with sequence similarity 64, member A 2.38 Prune2 protein prune homolog 2 2.34 Ccnb2 G2/mitotic-specific cyclin-B2 2.32 Aspm abnormal spindle-like microcephaly-associated protein homolog 2.32 Prelid2 PRELI domain-containing protein 2 2.29 Sncaip synuclein, alpha interacting protein (synphilin), isoform CRA_b 2.28 Tmem71 transmembrane protein 71 2.28 Mmp9 matrix metalloproteinase-9 precursor 2.28 Kif15 kinesin-like protein KIF15 2.28 Ccl6 C-C motif chemokine 6 precursor 2.24 Cxcl3 C-X-C motif chemokine 3 precursor 2.20 1-Mar E3 ubiquitin-protein ligase MARCH1 isoform 3 2.20 Il33 interleukin-33 precursor 2.20 Fam36a Protein FAM36A 2.20 Scd2 acyl-CoA desaturase 2 2.19 Snhg3 mCG118858 2.17 Kif20b kinesin-like protein KIF20B 2.16 Pkd2l2 polycystic kidney disease 2-like 2 protein 2.16 Psg22 mCG3735 2.16 Mis18bp1 expressed sequence C79407 2.15 Cldn2 claudin-2 2.15 8430408G22Rik protein DEPP 2.15 Hells lymphocyte-specific helicase 2.14 Casc5 protein CASC5 2.13 Myh15 myosin-15 2.12 Kctd12 BTB/POZ domain-containing protein KCTD12 2.10 Csf3r granulocyte colony-stimulating factor receptor precursor 2.08 2810417H13Rik mCG131663, isoform CRA_b 2.06 Cybb cytochrome b-245 heavy chain 2.06 Clec1a C-type lectin domain family 1 member A 2.06 Ect2 mKIAA4037 protein 2.04 C330027C09Rik RIKEN cDNA C330027C09, isoform CRA_a 2.03 Sgol1 shugoshin-like 1 2.02 C030044B11Rik mCG1043891 2.01 Rassf4 Ras association (RalGDS/AF-6) domain family member 4 2.00 St8sia4 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4 2.00 Efha2 EF-hand domain-containing family member A2 1.99 Prr11 proline-rich protein 11 1.98 Fam83b Protein FAM83B 1.97 Clec4n C-type lectin domain family 6 member A isoform 1 1.97 Enpp2 ectonucleotide pyrophosphatase/phosphodiesterase family member 2 isoform 1 1.97 Trp53i11 Trp53 inducible protein 11, isoform CRA_d 1.94 Prrg1 proline rich Gla (G-carboxyglutamic acid) 1 1.94 5830433M19Rik likely ortholog of H. sapiens chromosome 9 open reading frame 82 (C9orf82) 1.93 Cdca2 cell division cycle-associated protein 2 1.92 Cd14 monocyte differentiation antigen CD14 precursor 1.92 Col4a3 collagen alpha-3(IV) chain precursor 1.90 Gpr84 G-protein coupled receptor 84 1.90 Ccl4 C-C motif chemokine 4 1.89 Sorcs1 VPS10 domain-containing receptor SorCS1 1.89 Cenpf centromere protein F 1.89 Fam83d mCG15735 1.88 Bub1 budding uninhibited by benzimidazoles 1 homolog (S. cerevisiae), isoform CRA_a 1.87 Kif14 kinesin-like protein KIF14 1.86 Fam132b Protein FAM132B 1.85 Muc1 mucin-1 1.85 Plch1 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase eta-1 isoform 2 1.85 Ccdc15 coiled-coil domain-containing protein 15 1.84 Ube2c ubiquitin-conjugating enzyme E2 C 1.84 Cxcr4 C-X-C chemokine receptor type 4 1.82 Igfbp3 insulin-like growth factor-binding protein 3 1.82 Clec7a C-type lectin domain family 7 member A 1.81 Lcp2 lymphocyte cytosolic protein 2 1.80 Hmmr hyaluronan mediated motility receptor 1.79 Kif11 kinesin family member 11, isoform CRA_b 1.79 Spic transcription factor Spi-C 1.79 Lrch2 leucine-rich repeats and calponin homology (CH) domain containing 2 1.78 Gulp1 PTB domain-containing engulfment adapter protein 1 1.78 Fam199x cDNA sequence BC031748 1.78 Tpx2 TPX2, microtubule-associated protein homolog (Xenopus laevis), isoform CRA_b 1.77 Ms4a6d membrane-spanning 4-domains subfamily A member 6D 1.76 Ccl7 C-C motif chemokine 7 1.75 Kif23 kinesin family member 23 1.74 Ptpn4 tyrosine-protein phosphatase non-receptor type 4 1.73 Abcc9 ATP-binding cassette sub-family C member 9 isoform a 1.72 Sorl1 sortilin-related receptor 1.72 Pbk lymphokine-activated killer T-cell-originated protein kinase 1.71 Mastl microtubule-associated serine/threonine-protein kinase-like 1.70 Ehf ets homologous factor, isoform CRA_d 1.70 Mybl1 myeloblastosis oncogene-like 1, isoform CRA_a 1.69 Gen1 flap endonuclease GEN homolog 1 1.69 Dock11 dedicator of cytokinesis protein 11 1.68 Fam169a Fam169a protein 1.67 Cep55 centrosomal protein 55, isoform CRA_d 1.67 Col4a4 collagen alpha-4(IV) chain precursor 1.66 Sass6 spindle assembly abnormal protein 6 homolog 1.65 Ndc80 kinetochore protein NDC80 homolog 1.65 Eya4 eyes absent homolog 4 1.64 Kcne3 potassium voltage-gated channel subfamily E member 3 1.64 C1galt1 glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1 1.64 Vash1 vasohibin-1 1.64 Idi1 isopentenyl-diphosphate Delta-isomerase 1 1.63 Plek pleckstrin 1.62 Insig1 insulin-induced gene 1 protein 1.62 Elovl6 elongation of very long chain fatty acids protein 6 1.62 Cp ceruloplasmin, isoform CRA_c 1.61 Lyve1 lymphatic vessel endothelial hyaluronic acid receptor 1 precursor 1.61 Marco macrophage receptor MARCO 1.61 Ccna2 cyclin-A2 1.60 Mki67 antigen KI-67 1.60 Siglece sialic acid binding Ig-like lectin E 1.60 Prpf40a pre-mRNA-processing factor 40 homolog A 1.59 Ncapg non-SMC condensin I complex, subunit G 1.58 Chm rab proteins geranylgeranyltransferase component A 1 1.58 Slx1b GIY-YIG domain containing 2, isoform CRA_d 1.57 Lin9 protein lin-9 homolog isoform 1 1.57 Anln anillin, actin binding protein (scraps homolog, Drosophila), isoform CRA_b 1.57 Utp14b U3 small nucleolar RNA-associated protein 14 homolog B 1.56 Acin1 apoptotic chromatin condensation inducer in the nucleus isoform 4 1.56 Kif20a kinesin family member 20A, isoform CRA_b 1.55 Prss22 brain-specific serine protease 4 1.55 Dram1 DNA damage-regulated autophagy modulator protein 1 1.55 Emr1 EGF-like module containing, mucin-like, hormone receptor-like sequence 1, isoform CRA_c 1.55 Slc7a11 cystine/glutamate transporter 1.54 Ctsc dipeptidyl peptidase 1 preproprotein 1.54 Gsta3 glutathione S-transferase A3 1.54 Ttk Ttk protein kinase, isoform CRA_c 1.54 Zdhhc2 palmitoyltransferase ZDHHC2 1.54 Mfap3l microfibrillar-associated protein 3-like, isoform CRA_b 1.54 Zcchc10 zinc finger, CCHC domain containing 10, isoform CRA_b 1.54 Ccbe1 collagen and calcium binding EGF domains 1, isoform CRA_e 1.53 Hsd17b7 3-keto-steroid reductase 1.53 Rp2h protein XRP2 1.52 Pcmtd1 rCG30411, isoform CRA_b [Rattus norvegicus] 1.51 Nbeal1 neurobeachin like 1 1.51 Msr1 macrophage scavenger receptor types I and II isoform a 1.51 Cmah cytidine monophospho-N-acetylneuraminic acid hydroxylase, isoform CRA_c 1.50 Trim59 tripartite motif-containing protein 59 1.50 Itga2 integrin alpha-2 precursor 1.49 Lpar4 lysophosphatidic acid receptor 4 [Rattus norvegicus] 1.49 Smc2 structural maintenance of chromosomes protein 2 1.49 Il1rn interleukin-1 receptor antagonist protein isoform 2 1.48 Atad2 ATPase family AAA domain-containing protein 2 1.48 Cav2 caveolin-2 1.48 Acsl3 long-chain-fatty-acid--CoA ligase 3 isoform a 1.47 Cep290 centrosomal protein of 290 kDa 1.47 Ccdc88a girdin 1.46 Clec4e C-type lectin domain family 4 member E 1.46 Scd1 acyl-CoA desaturase 1 1.45 F8 coagulation factor VIII isoform 3 1.45 Pak3 serine/threonine-protein kinase PAK 3 isoform C 1.45 Vma21 vacuolar ATPase assembly integral membrane protein VMA21 1.45 Aurka serine/threonine-protein kinase 6 1.43 Pla2g7 platelet-activating factor acetylhydrolase 1.43 Slc4a7 sodium bicarbonate cotransporter 3 1.43 Fam46a mCG11933 1.43 Ckap2l cytoskeleton associated protein 2-like, isoform CRA_a 1.42 Rnf125 E3 ubiquitin-protein ligase RNF125 1.42 Yes1 Yamaguchi sarcoma viral (v-yes) oncogene homolog 1, isoform CRA_b 1.42 Wisp1 WNT1 inducible signaling pathway protein 1, isoform CRA_d 1.41 Rnf122 ring finger protein 122, isoform CRA_b 1.41 Tspan2 tetraspanin-2 1.41 D19Ertd737e DNA segment, Chr 19, ERATO Doi 737, expressed, isoform CRA_d 1.41 Galnt3 polypeptide N-acetylgalactosaminyltransferase 3 1.41 Tmtc3 transmembrane and TPR repeat-containing protein 3 isoform 2 1.40 Marcks myristoylated alanine-rich C-kinase substrate [Bos taurus] 1.40 Stk3 serine/threonine-protein kinase 3 1.40 Anxa13 annexin A13 1.39 Adamts4 A disintegrin and metalloproteinase with thrombospondin motifs 4 1.39 Abca5 ATP-binding cassette sub-family A member 5 1.39 Prc1 protein regulator of cytokinesis 1, isoform CRA_d 1.39 Fst follistatin 1.39 Treml4 triggering receptor expressed on myeloid cells-like 4, isoform CRA_c 1.38 Zfp800 zinc finger protein 800 1.38 Ccnc cyclin C, isoform CRA_d 1.38 Top2a DNA topoisomerase 2-alpha 1.38 Myo1b myosin-Ib isoform 2 1.38 Hmgn5 GARP45 1.38 Rb1 retinoblastoma-associated protein 1.37 Atp11c probable phospholipid-transporting ATPase 11C isoform a 1.37 Lcorl ligand-dependent nuclear receptor corepressor-like protein isoform 1 1.37 Kif4 chromosome-associated kinesin KIF4 1.36 Rad51ap1 RAD51-associated protein 1 1.36 AA467197 normal mucosa of esophagus-specific gene 1 protein 1.36 Gtpbp10 GTP-binding protein 10 1.36 Tmx3 mKIAA1830 protein 1.35 Birc5 baculoviral IAP repeat-containing protein 5 isoform 1 1.35 Aldh1a2 retinal dehydrogenase 2 1.35 Usp45 ubiquitin carboxyl-terminal hydrolase 45 1.34 Iqgap3 IQ motif containing GTPase activating protein 3 1.34 7-Mar E3 ubiquitin-protein ligase MARCH7 1.34 Dnajb9 DnaJ (Hsp40) homolog, subfamily B, member 9 1.34 Chek1 serine/threonine-protein kinase Chk1 1.32 Smc4 structural maintenance of chromosomes protein 4 1.32 Hif1a hypoxia inducible factor 1, alpha subunit, isoform CRA_b 1.32 Ccdc34 coiled-coil domain-containing protein 34 1.31 Spp1 secreted phosphoprotein 1, isoform CRA_d 1.31 Gmnn geminin 1.31 Prkcb protein kinase C beta type 1.31 Prkaa2 5'-AMP-activated protein kinase catalytic subunit alpha-2 1.30 Spc25 spindle pole body component 25 homolog (S. cerevisiae), isoform CRA_b 1.30 Fam117a RIKEN cDNA 5730593F17 1.30 Rab11fip2 Rab11-family interacting protein 2 isoform 1 1.30 Cdc20 cell division cycle protein 20 homolog 1.30 Lysmd3 lysM and putative peptidoglycan-binding domain-containing protein 3 1.29 Fmr1 fragile X mental retardation protein 1 homolog 1.29 Manea glycoprotein endo-alpha-1,2-mannosidase 1.29 Lrrcc1 mKIAA1764 protein 1.29 Vkorc1l1 vitamin K epoxide reductase complex subunit 1-like protein 1 isoform 2 1.29 Zfp518a zinc finger protein 518A 1.28 Gm11428 mCG55370 1.28 Fyb FYN-binding protein 1.28 Fsd1l FSD1-like protein isoform 1 1.28 F630043A04Rik RIKEN cDNA F630043A04, isoform CRA_b 1.28 Fam101b family with sequence similarity 101, member B 1.28 Mzt1 mCG1042562 1.27 Homer1 homer protein homolog 1 isoform L 1.26 Gng2 mCG144516 1.26 Gpr126 G-protein coupled receptor 126 precursor 1.26 Psd3 PH and SEC7 domain-containing protein 3 isoform 2 1.26 Vamp7 vesicle-associated membrane protein 7 1.26 Nid2 nidogen-2 precursor 1.26 Cpeb4 cytoplasmic polyadenylation element-binding protein 4 1.26 Txlng gamma-taxilin 1.26 Snrnp27 U4/U6.U5 small nuclear ribonucleoprotein 27 kDa protein 1.25 Tmed5 Transmembrane emp24 protein transport domain containing 5 1.25 Acsl4 acyl-CoA synthetase long-chain family member 4, isoform CRA_b 1.25 Amot Angiomotin 1.25 Dcdc2a mKIAA1154 protein 1.25 Upf3b regulator of nonsense transcripts 3B 1.24 Plcb4 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-4 1.24 Plod2 procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 isoform 1 1.24 Hs6st2 Heparan-sulfate 6-O-sulfotransferase 2 1.24 Pcdhb16 protocadherin beta 16 1.24 Mme membrane metallo endopeptidase, isoform CRA_a 1.24 Apob apolipoprotein B precursor 1.24 Pde3b cGMP-inhibited 3',5'-cyclic phosphodiesterase B 1.24 Pik3c2a phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing subunit alpha 1.23 Jhdm1d lysine-specific demethylase 7 1.22 Slc12a2 solute carrier family 12 member 2 1.22 Uevld ubiquitin-conjugating enzyme E2 variant 3 1.22 C5ar1 C5a anaphylatoxin chemotactic receptor 1.22 Filip1 filamin-A-interacting protein 1 1.22 Itgam integrin alpha-M isoform 2 precursor 1.22 Suz12 suppressor of zeste 12 homolog (Drosophila), isoform CRA_c 1.22 Cacybp calcyclin binding protein 1.21 Naa50 N-acetyltransferase 13, isoform CRA_c 1.21 A630089N07Rik PREDICTED: putative transposase element L1Md-A101/L1Md-A102/L1Md-A2-like 1.21 Pcmtd2 mKIAA3003 protein 1.21 Cyp26b1 cytochrome P450, family 26, subfamily b, polypeptide 1, isoform CRA_b 1.20 Chml rab proteins geranylgeranyltransferase component A 2 1.20 Scoc short coiled-coil protein isoform a 1.20 Fcgr3 low affinity immunoglobulin gamma Fc region receptor III 1.20 Tnf tumor necrosis factor 1.20 A230046K03Rik WASH complex subunit 7 1.20 Eea1 early endosome antigen 1 1.20 Rrm2 ribonucleotide reductase M2 1.20 Sgol2 shugoshin-like 2 isoform 2 1.20 D730040F13Rik likely ortholog of H. sapiens chromosome 9 open reading frame 5 (C9orf5) 1.20 Styx serine/threonine/tyrosine-interacting protein 1.19 Brca2 breast cancer 2 1.18 Atrx transcriptional regulator ATRX 1.18 Tceal8 transcription elongation factor A protein-like 8 1.18 Rock2 rho-associated protein kinase 2 1.18 Dtx4 protein deltex-4 1.18 Zfp36l1 zinc finger protein 36, C3H1 type-like 1 1.17 Cd44 CD44 antigen isoform a precursor 1.17 Cdk1 cyclin-dependent kinase 1 1.17 Adamts5 A disintegrin and metalloproteinase with thrombospondin motifs 5 precursor 1.17 Fam49a DNA segment, Chr 12, ERATO Doi 553, expressed, isoform CRA_b 1.17 Kif2c kinesin-like protein KIF2C 1.17 Il1rap interleukin-1 receptor accessory protein isoform d precursor 1.17 Olfml2b olfactomedin-like protein 2B precursor 1.16 Ammecr1 PREDICTED: AMME syndrome candidate gene 1 protein homolog 1.16 Rbm34 RNA-binding protein 34 1.16 Dbf4 protein DBF4 homolog A isoform 1 1.16 Col4a5 procollagen type IV alpha 5 1.15 Sc4mol C-4 methylsterol oxidase 1.15 Dzip3 E3 ubiquitin-protein ligase DZIP3 isoform 1 1.15 Cul4b cullin 4B 1.15 Psip1 PC4 and SFRS1-interacting protein 1.15 Espl1 extra spindle poles-like 1 (S. cerevisiae), isoform CRA_b 1.15 Ube2g1 ubiquitin-conjugating enzyme E2G 1 (UBC7 homolog, C. elegans), isoform CRA_c 1.15 Fnip1 folliculin-interacting protein 1 1.15 Osbpl8 oxysterol-binding protein-like protein 8 isoform a 1.14 Tbca tubulin-specific chaperone A 1.14 Itgav integrin alpha-V precursor 1.14 Klhl4 kelch-like 4 (Drosophila) 1.14 Dlgap5 discs, large homolog 7 (Drosophila), isoform CRA_a 1.13 Xrn1 5'-3' exoribonuclease 1 1.13 Pou2f1 POU domain, class 2, transcription factor 1 isoform B 1.13 Rif1 telomere-associated protein RIF1 1.13 Sp4 transcription factor Sp4 isoform 2 1.13 Man2a1 mannosidase 2, alpha 1 1.13 Arhgap18 Rho GTPase activating protein 18 1.13 Rg9mtd2 RNA (guanine-9-)-methyltransferase domain-containing protein 2 1.13 Atp7a ATPase, Cu++ transporting, alpha polypeptide, isoform CRA_b 1.13 Tnfsf10 tumor necrosis factor ligand superfamily member 10 1.13 Mad2l1 mCG125314, isoform CRA_b 1.12 Zbtb41 zinc finger and BTB domain-containing protein 41 1.12 A630007B06Rik oocyte-testis gene 1 1.12 Taf1d TATA box-binding protein-associated factor RNA polymerase I subunit D isoform 2 1.12 Calcrl calcitonin gene-related peptide type 1 receptor precursor 1.12 Pter phosphotriesterase related, isoform CRA_a 1.11 Fam38b piezo2 1.11 Mob1b mps one binder kinase activator-like 1A 1.11 Mndal myeloid cell nuclear differentiation antigen-like protein 1.11 Rb1cc1 RB1-inducible coiled-coil protein 1 1.11 4933421E11Rik receptor-interacting factor 1 isoform 1 1.10 Fam63b RIKEN cDNA B230380D07, isoform CRA_d 1.10 Zfp644 zinc finger protein 644 1.10 Acer3 alkaline ceramidase 3 1.10 Olr1 oxidized low density lipoprotein (lectin-like) receptor 1 1.10 Map3k2 mitogen-activated protein kinase kinase kinase 2 1.10 Esco1 N-acetyltransferase ESCO1 1.10 Zbed6 zinc finger BED domain-containing protein 6 1.10 Hook3 protein Hook homolog 3 1.10 Cldn1 claudin-1 1.10 Ankrd32 ankyrin repeat domain-containing protein 32 1.09 Sarnp SAP domain-containing ribonucleoprotein 1.09 Steap2 metalloreductase STEAP2 1.09 Hmgb2 high mobility group protein B2 1.09 Dhx36 probable ATP-dependent RNA helicase DHX36 1.09 Ccdc99 protein Spindly 1.09 Casp3 caspase-3 1.09 2700089E24Rik mCG1039243, isoform CRA_c 1.09 Lin7c protein lin-7 homolog C 1.09 Scai protein SCAI 1.09 Ell2 RNA polymerase II elongation factor ELL2 1.09 Arhgap11a mKIAA0013 protein 1.08 Gm3414 Putative uncharacterized protein 1.08 Fktn fukutin 1.08 Slfn9 schlafen 9 1.08 3110003A17Rik 3110003A17Rik protein 1.08 Stk17b serine/threonine kinase 17b (apoptosis-inducing), isoform CRA_b 1.07 Tab3 mKIAA4135 protein 1.07 Tanc2 protein TANC2 1.07 Ube2w mCG13474 1.07 Abca1 ATP-binding cassette sub-family A member 1 1.07 1810029B16Rik UPF0534 protein C4 1.07 Pdcd10 programmed cell death protein 10 1.07 Zfp91 E3 ubiquitin-protein ligase ZFP91 1.06 Ggps1 geranylgeranyl pyrophosphate synthase 1.06 Polr3g DNA-directed RNA polymerase III subunit RPC7 1.06 Ptprc protein tyrosine phosphatase, receptor type, C, isoform CRA_b 1.06 7-Sep Septin-7 1.06 Serpinb9 serine (or cysteine) proteinase inhibitor, clade B, member 9 1.06 Itga8 integrin alpha-8 precursor 1.06 Trpm7 transient receptor potential cation channel subfamily M member 7 isoform 2 1.05 Trip11 thyroid hormone receptor interactor 11 1.05 Fignl1 fidgetin-like 1, isoform CRA_b 1.05 Exo1 exonuclease 1 1.04 Dennd1b DENN domain-containing protein 1B isoform 1 1.04 Hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1, isoform CRA_b 1.04 Fam76b Protein FAM76B 1.04 Zbtb10 zinc finger and BTB domain containing 10 1.04 Dr1 hypothetical protein PANDA_006355 1.04 Naa16 N-alpha-acetyltransferase 16, NatA auxiliary subunit 1.04 Lnpep leucyl-cystinyl aminopeptidase 1.04 Mier3 mesoderm induction early response protein 3 1.03 Cenpc1 centromere protein C 1 1.03 Arhgap5 rho GTPase-activating protein 5 1.03 Ncoa7 nuclear receptor coactivator 7 isoform 2 1.03 Cd164 sialomucin core protein 24 1.03 Malat1 mCG1042565, isoform CRA_a 1.03 Pmp22 Peripheral myelin protein 22 1.03 Ugcg ceramide glucosyltransferase 1.03 Sc5d lathosterol oxidase 1.02 Tfb2m dimethyladenosine transferase 2, mitochondrial 1.02 Ncoa6 nuclear receptor coactivator 6 1.02 Cxcl1 Cxcl1 protein 1.02 Kcnj2 inward rectifier potassium channel 2 1.02 Prex2 phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 protein isoform 1 1.02 Sh3kbp1 SH3 domain-containing kinase-binding protein 1 isoform 2 1.02 Prom1 prominin-1 isoform s8 1.01 Klf6 Krueppel-like factor 6 1.01 Gda mKIAA1258 protein 1.01 Phf17 mKIAA1807 protein 1.01 Blm Bloom syndrome protein homolog isoform 1 1.01 Arid4b AT-rich interactive domain-containing protein 4B isoform 1 1.01 Cd2ap CD2-associated protein 1.01 E2f7 transcription factor E2F7 1.01 Rnpc3 RNA-binding protein 40 1.00 Mgll monoglyceride lipase isoform d 1.00 Atad5 ATPase family AAA domain-containing protein 5 1.00 Prrg4 transmembrane gamma-carboxyglutamic acid protein 4 precursor 1.00 4921524J17Rik UPF0547 protein C16orf87 homolog 1.00 Apobec3 DNA dC->dU-editing enzyme APOBEC-3 isoform 1 1.00 Pdk2 pyruvate dehydrogenase kinase, isoenzyme 2 precursor -1.00 Plxna4 plexin-A4 precursor -1.00 Abi3 phosphoethanolamine/phosphocholine phosphatase [Rattus norvegicus] -1.00 Pnkp polynucleotide kinase 3'- phosphatase, isoform CRA_d -1.01 Zfpm1 zinc finger protein ZFPM1 -1.01 Pdrg1 p53 and DNA damage-regulated protein 1 -1.02 C1qb complement C1q subcomponent subunit B precursor -1.02 Fbn1 fibrillin-1 -1.02 Xdh xanthine dehydrogenase, isoform CRA_a -1.02 Rrp9 U3 small nucleolar RNA-interacting protein 2 -1.02 Cpt2 carnitine O-palmitoyltransferase 2, mitochondrial precursor -1.03 Shroom1 protein Shroom1 -1.04 Prmt2 protein arginine N-methyltransferase 2 -1.04 Polr2i mCG22814 -1.04 Stk40 serine/threonine kinase 40, isoform CRA_b -1.04 Bst2 bone marrow stromal cell antigen 2, isoform CRA_b -1.05 Ccs copper chaperone for superoxide dismutase -1.05 Fbxw4 F-box/WD repeat-containing protein 4 -1.05 Dmpk myotonin-protein kinase isoform 2 -1.06 Fam203a brain protein 16 -1.06 Cd5l CD5 antigen-like precursor -1.06 Telo2 RIKEN cDNA 1200003M09, isoform CRA_c -1.06 Xaf1 XIAP-associated factor 1 -1.06 Slc25a19 mitochondrial thiamine pyrophosphate carrier -1.07 0910001L09Rik mCG7441, isoform CRA_a -1.07 Cebpa CCAAT/enhancer-binding protein alpha -1.07 Mib2 E3 ubiquitin-protein ligase MIB2 -1.08 Eng endoglin isoform 2 -1.08 Nenf neudesin precursor -1.08 Tmem204 transmembrane protein 204 -1.08 Eppk1 epiplakin -1.09 Calhm2 RIKEN cDNA 2810048G17, isoform CRA_a -1.09 Rspo3 R-spondin-3 precursor -1.09 H2-D1 H-2 class I histocompatibility antigen, L-D alpha chain precursor -1.10 Atf5 cyclic AMP-dependent transcription factor ATF-5 -1.10 Rapgef3 rap guanine nucleotide exchange factor 3 isoform 3 -1.10 Snai1 zinc finger protein SNAI1 -1.10 Fhdc1 mKIAA1727 protein -1.11 Nr1d1 nuclear receptor subfamily 1 group D member 1 -1.11 Ndufs7 NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial precursor -1.12 Tap1 antigen peptide transporter 1 isoform 2 -1.12 Krt8 keratin, type II cytoskeletal 8 -1.13 Hspb1 heat shock protein beta-1 -1.13 Mrps12 28S ribosomal protein S12, mitochondrial precursor -1.14 Mad2l1bp mKIAA0110 protein -1.14 Abcc10 multidrug resistance-associated protein 7 isoform mrp7B -1.14 Rinl RIKEN cDNA 5830482F20, isoform CRA_a -1.15 Twf2 twinfilin-2 -1.15 Id1 helix-loop-helix protein (Id) -1.16 Gata4 transcription factor GATA-4 -1.16 Igsf9 immunoglobulin superfamily, member 9, isoform CRA_a -1.17 Acads short-chain specific acyl-CoA dehydrogenase, mitochondrial precursor -1.17 Alb serum albumin precursor -1.17 Pknox2 homeobox protein PKNOX2 -1.18 Nbl1 neuroblastoma suppressor of tumorigenicity 1 precursor -1.19 Armc6 mCG23104, isoform CRA_b -1.20 Mettl11a alpha N-terminal protein methyltransferase 1A -1.20 Pcyox1l prenylcysteine oxidase-like precursor -1.22 Htra1 serine protease HTRA1 precursor -1.22 Zfp59 zinc finger protein 59 -1.24 Tfeb transcription factor EB, isoform CRA_c -1.25 Hint2 histidine triad nucleotide-binding protein 2, mitochondrial precursor -1.26 Rhod rho-related GTP-binding protein RhoD precursor -1.27 Bmp6 bone morphogenetic protein 6, isoform CRA_a -1.27 Ltbp4 latent-transforming growth factor beta-binding protein 4 isoform b -1.28 Neat1 mCG148129 -1.29 Adcy1 adenylate cyclase type 1 -1.30 Fam171a2 Protein FAM171A2 -1.31 AU021092 UPF0764 protein C16orf89 homolog -1.32 Entpd1 ectonucleoside triphosphate diphosphohydrolase 1, isoform CRA_b -1.35 Ehd3 EH domain-containing protein 3 -1.36 Cspg4 chondroitin sulfate proteoglycan 4 precursor -1.37 Ccdc48 coiled-coil domain-containing protein 48 -1.39 Hs3st1 heparan sulfate glucosamine 3-O-sulfotransferase 1 precursor -1.42 Vwf Von Willebrand factor homolog -1.43 Ceacam1 carcinoembryonic antigen-related cell adhesion molecule 1 isoform 1 -1.44 Psmg4 proteasome assembly chaperone 4 isoform b -1.46 Il18bp interleukin-18-binding protein precursor -1.46 H2-T23 H-2 class I histocompatibility antigen, D-37 alpha chain precursor -1.47 Smad7 MAD homolog 7 (Drosophila), isoform CRA_a -1.47 5430435G22Rik RIKEN cDNA 5430435G22, isoform CRA_a -1.50 Flnc filamin-C -1.50 Smarcd3 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 3 -1.51 Oas1a 2'-5'-oligoadenylate synthase 1A -1.52 Ace angiotensin-converting enzyme isoform 1 -1.54 Lgals3 Galectin-3 -1.55 Smad6 mothers against decapentaplegic homolog 6 -1.57 E4f1 transcription factor E4F1 -1.57 Adap2 arf-GAP with dual PH domain-containing protein 2 -1.59 Snta1 syntrophin, acidic 1, isoform CRA_a -1.61 Boc brother of CDO precursor -1.62 Krt17 keratin, type I cytoskeletal 17 -1.62 Trem2 TREM2B splice variant -1.63 Id3 DNA-binding protein inhibitor ID-3 -1.67 H2-Q8 H-2 class I histocompatibility antigen, Q8 alpha chain -1.68 Dpm3 dolichol-phosphate mannosyltransferase subunit 3 -1.71 Efhd1 EF hand domain containing 1, isoform CRA_c -1.71 Bmper BMP-binding endothelial regulator protein precursor -1.71 Gja4 gap junction alpha-4 protein -1.71 Pf4 platelet factor 4 precursor -1.71 Zfp862 zinc finger protein 862-like -1.71 Gata2 endothelial transcription factor GATA-2 -1.72 Zfp775 zinc finger protein 775 -1.74 3000002C10Rik Glyceraldehyde-3-phosphate dehydrogenase -1.75 Cnn1 calponin-1 -1.78 Tgfbi transforming growth factor-beta-induced protein ig-h3 precursor -1.78 Lims2 LIM and senescent cell antigen-like-containing domain protein 2 -1.81 H2-Q6 histocompatibility 2, Q region locus 6 -1.82 Cd74 H-2 class II histocompatibility antigen gamma chain isoform 2 -1.83 Cd300lb CMRF35-like molecule 7 -1.84 Rasl11b ras-like protein family member 11B -1.84 Rn7sk zinc finger CCCH-type domain containing 3 -1.86 Adamts2 A disintegrin and metalloproteinase with thrombospondin motifs 2 -1.87 H2-Ab1 histocompatibility 2, class II antigen A, beta 1 precursor -1.89 Nudt7 nudix (nucleoside diphosphate linked moiety X)-type motif 7, isoform CRA_b -1.90 Nr4a1 nuclear receptor subfamily 4 group A member 1 -1.96 Pltp phospholipid transfer protein precursor -1.97 Tmem200b transmembrane protein 200B [Homo sapiens] -2.05 Atoh8 protein atonal homolog 8 -2.08 Plac8 placenta-specific gene 8 protein -2.12 Zbp1 Z-DNA-binding protein 1 isoform 2 -2.17 Vsig4 V-set and immunoglobulin domain-containing protein 4 -2.34 H2-Aa histocompatibility 2, class II antigen A, alpha precursor -2.44 Ly6a lymphocyte antigen 6 complex, locus A, isoform CRA_b -2.44 Klhdc8a kelch domain containing 8A, isoform CRA_b -2.56 Igf2 insulin-like growth factor II isoform 1 preproprotein -2.75 Cyp1a1 cytochrome P450 1A1 isoform 1 -2.90 Irf7 interferon regulatory factor 7 -2.97 C4b complement C4-B precursor -3.21 Foxn1 forkhead box protein N1 -3.21 Pdlim3 PDZ and LIM domain protein 3 -3.37 Dio3 type III iodothyronine deiodinase -4.44 Blk B lymphoid kinase -10.34