Highly Activated Cytotoxic CD8 T Cells Express Protective IL-10 at the Peak of Coronavirus-Induced Encephalitis

This information is current as Kathryn Trandem, Jingxian Zhao, Erica Fleming and Stanley of September 28, 2021. Perlman J Immunol 2011; 186:3642-3652; Prepublished online 11 February 2011; doi: 10.4049/jimmunol.1003292

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Highly Activated Cytotoxic CD8 T Cells Express Protective IL-10 at the Peak of Coronavirus-Induced Encephalitis

Kathryn Trandem,* Jingxian Zhao,†,‡ Erica Fleming,† and Stanley Perlman*,†

Acute viral encephalitis requires rapid pathogen elimination without significant bystander tissue damage. In this article, we show that IL-10, a potent anti-inflammatory , is produced transiently at the peak of infection by CD8 T cells in the brains of coronavirus-infected mice. IL-10+CD8 and IL-102CD8 T cells interconvert during acute disease, possibly based on recent Ag exposure. Strikingly, IL-10+CD8 T cells were more highly activated and cytolytic than IL-102CD8 T cells, expressing greater levels of proinflammatory and , as well as cytotoxic . Even though these cells are highly proin- flammatory, IL-10 expressed by these cells was functional. Furthermore, IL-10 produced by CD8 T cells diminished disease severity in mice with coronavirus-induced acute encephalitis, suggesting a self-regulatory mechanism that minimizes immuno-

pathological changes. The Journal of Immunology, 2011, 186: 3642–3652. Downloaded from

nterleukin-10 is a potent anti-inflammatory cytokine with during chronic infections, only recently has its role in non- a crucial role in limiting proinflammatory responses and persisting virus infections begun to be appreciated. Unlike chronic I preventing autoimmune diseases. Although several factors infections, CD8 T cells, in addition to CD4 T cells, have been contribute to the anti-inflammatory repertoire, the role of IL-10 is identified as major producers of IL-10 in experimental respira- nonredundant. Thus, a spontaneous enterocolitis occurs in mice tory infections caused by influenza A virus, SV5, and respiratory http://www.jimmunol.org/ that are deficient in IL-10, because of florid proinflammatory syncytial virus (10–13); little is known about IL-10 expression by responses to normal bacterial flora (1). In addition, a deficiency in CD8 T cells. IL-10 results in exaggerated proinflammatory responses during Control of the pathogen and consequent inflammatory response bacterial, protozoal, and viral infections (2, 3). In most of these is especially important in infections of the CNS, including viral studies, IL-10 is produced by CD4 T cells (including effector and encephalitis, to minimize tissue damage. Therefore, to investigate regulatory T cells) or by or dendritic cells, although the role of IL-10 in acute encephalitis, in particular IL-10 expressed NK cells have also been identified as an important source in by CD8 T cells, we infected mice with the neurotropic JHM strain systemic Toxoplasma gondii infections (2, 4, 5). Production of of mouse hepatitis virus (14, 15). The J2.2-V-1 variant of this virus IL-10 by IFN-g+CD4 (Th1) T cells is dependent on strong Ag produces encephalomyelitis, with progression to a chronic de- by guest on September 28, 2021 stimulation; in vitro, these cells required greater dosages of pep- myelinating disease. Infectious virus is cleared by 10–14 d post- tide to express maximal levels of IL-10 compared with IFN-g (6). infection (p.i.), although viral Ag and RNA can be detected for In addition, IL-10 expression in CD4 T cells is dependent on months after infection accompanied by low levels of demyeli- stimulation via IL-12 and STAT-4 signaling, and requires activa- nation (14, 16). Inflammation is maximal and extensive through- tion of ERK-dependent pathways. out the acute phase of the infection (14). IL-10 production before complete pathogen clearance contrib- In this article, we demonstrate that expression of IL-10 by CD8 utes to persistent infection. Neutralization of IL-10 in persistent T cells during peak inflammation is not limited to respiratory leishmania or lymphocytic choriomeningitis virus infections re- infections, and that ∼50% of IL-10–producing cells in the infected sults in pathogen clearance, with concomitant immunopatholo- brain are murine hepatitis virus-specific cytotoxic CD8 T cells. In gical disease in some instances (7–9). Although it is clear that addition, this CD8 T cell-derived IL-10 ameliorates clinical disease IL-10 has an important role in prolonging pathogen persistence in acute encephalitis and subsequent demyelination. IL-10 ex- pression by CD8 T cells requires strong Ag stimulation and sig- *Interdisciplinary Program in Immunology, University of Iowa, Iowa City, IA 52242; naling through MAPK pathways. Furthermore, using microarray †Department of Microbiology, University of Iowa, Iowa City, IA 52242; and profiling, quantitative RT-PCR (qRT-PCR), and analyses, ‡ Institute for Tissue Transplantation and Immunology, Jinan University, Guangzhou we demonstrate that the fraction of CD8 T cells that produce IL-10 510630, China are hyperactivated CD8 T cells. Thus, cytotoxic CD8 T cells may Received for publication October 6, 2010. Accepted for publication January 3, 2011. regulate their inflammatory effects during acute infections by pro- This work was supported by the National Institutes of Health (Grant NS36592 and Training Grant T32 AI007511-14 to K.T.) and the National Soci- ducing IL-10, and thereby minimize immunopathological disease. ety. The sequences presented in this article have been submitted to the Expression Materials and Methods Omnibus under accession number GSE25846. Mice Address correspondence and reprint requests to Dr. Stanley Perlman, Department of Specific pathogen-free 6-wk-old Thy1.2 and Thy1.1 C57BL/6 mice were Microbiology, BSB 3-730, University of Iowa, Iowa City, IA 52242. E-mail address: purchased from the National Institute (Bethesda, MD) and The [email protected] Jackson Laboratory (Bar Harbor, ME), respectively. Vert-X (C57BL/6 The online version of this article contains supplemental material. bicistronic IL-10/eGFP) mice were kindly provided by C. Karp (Cincin- 2/2 Abbreviations used in this article: CLN, cervical ; MFI, mean fluores- nati Children’s Hospital) (17). IL-10 mice (B6.129P2-Il10tm1Cgn/J) cence intensity; p.i., postinfection; qRT-PCR, quantitative RT-PCR. were bred at the University of Iowa. Mice were examined and weighed daily. All animal studies were approved by the University of Iowa Animal Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 Care and Use Committee. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003292 The Journal of Immunology 3643

Virus and viral titers T cells were positively selected or Thy1.2+ (CD8 and CD4) T cells were negatively selected using magnetic beads from STEMCELL Technologies Recombinant J2.2-V-1, used in all experiments, was propagated and titered (Vancouver, British Columbia, Canada), according to the manufacturer’s as described previously (18). Mice were inoculated intracerebrally with 500 protocol. GFP+, GFP2, CD69+, and CD692 CD8 and/or CD4 T cells were or 1500 PFU J2.2-V-1 in 30 ml DMEM. then sorted by a FACSDiva or a FACSAria (BD Biosciences). Postsort . Preparation of CNS mononuclear cells analysis showed that cells were 99% pure.

Brain mononuclear cells were isolated as described previously (16). In vitro transwell cell culture Abs and flow cytometric analyses Thy1.1 B6 lymph node cells were labeled with 2 mM CFSE, and 2 3 106 All Abs were purchased from BD Pharmingen (San Diego, CA) or eBio- cells were placed in the lower compartment of anti-CD3 (clone 145-2C11; science (San Diego, CA) unless indicated later. NK cells were CD45+CD32 eBioscience) coated wells. A 0.4-mm filter separated the two compart- NK1.1+. Macrophages were CD45+CD11b+Ly6C+Ly6G2. Neutrophils ments. Thy1.1 B6 splenocytes were pulsed with varying concentrations of were CD45+CD11b+Ly6C+Ly6G+. MHC class II was detected with anti–I- S510 peptide (1 mMto0mM) for 1 h before irradiation with 3000 rad. A total of 2 3 106 cells was placed in the upper compartment. Thy1.2 CD8+ A/anti–I-E. For detection of intracellular protein expression, CD8 or CD4 + + 2 3 4 T cells were stimulated with 1 mM peptide S510 or 5 mM peptide M133, GFP and CD8 GFP cells were sorted as described earlier, and 2.5 10 respectively, as previously described (16). IFN-g, IL-10, TNF, CD107a/b, cells were placed in the upper compartment with the irradiated spleno- and granzyme B were detected using Abs purchased from BioLegend (San cytes. A blocking anti–IL-10 mAb (clone JES5-2A5) or an isotype- Diego, CA). For detection of p-ERK, mAb clone D13.14.4E was pur- matched control mAb at 10 mg/ml (both from BioLegend) was added to chased from Technology (Beverly, MA). FcεR1 was de- some wells. Some cultures were treated with the MEK1/2 inhibitor PD tected using anti-DNP IgE (Sigma-Aldrich, St. Louis, MO) followed by rat 184161 (ERK1/2) or the p38 inhibitor PD169316 (Cayman Chemical, Ann Arbor, MI). For proliferation studies, lower compartment

anti-mouse IgE. M133-specific and S510-specific cells were also detected Downloaded from using PE-conjugated I-Ab/M133 and H-2Db/S510 tetramers, respectively, were stained with anti–CD4-PE and anti–CD8-PerCP, and examined by obtained from the National Institutes of Health Tetramer Core Facility flow cytometry for CFSE dilution after 48 h. Culture supernatants were (Atlanta, GA). Cells were analyzed using a FACSCalibur (BD Bio- harvested for cytokine detection by ELISA. sciences). In vivo GFP conversion Cell separation and sorting Thy1.2+GFP+ and Thy1.2+GFP2 (CD4 and CD8) cells from d7 J2.2-V-1–

Mononuclear cells were isolated from J2.2-V-1–infected Vert-X/Thy1.2 infected Vert-X mouse brains were sorted as described earlier. Either 1 3 http://www.jimmunol.org/ mice brains at day 7 p.i. Cells from three mice were pooled and CD8 106 GFP+ or 1 3 106 GFP2 cells were transferred in 300 ml PBS i.v. into by guest on September 28, 2021

FIGURE 1. IL-10 is produced by cells in the brain and not in the CLN or spleen. Vert-X mice were infected with 500 PFU J2.2-V-1. Brain, CLN, and spleen were harvested at various time points. A, FACS plots of cells stained for various surface markers together with GFP (IL-10) are shown at day 7 p.i. B–D, Frequency of GFP+ cells recovered from various cell types at stated time points in the brain (B), CLN (C), and spleen (D). Data are from three separate experiments at each time point with 12 mice/group. 3644 HYPERACTIVATED CYTOTOXIC CD8 T CELLS EXPRESS IL-10

J2.2-V-1–infected Thy1.1+ B6 mice at day 1 p.i. Six days later, lympho- Probe sets for exons were then summarized for a specific gene using the cytes were harvested, stained, and analyzed by flow cytometry. median value. After obtaining log2 expression values for , signifi- cance testing was performed comparing the two different CD8+ T cell + 2 ELISA groups (IL-10 versus IL-10 ). False discovery rate correction was applied to all of the p values to correct for multiple testing. Significance was J2.2-V-1–infected brains were weighed and homogenized directly into assessed by false discovery rate q , 0.25 and 2-fold change. Analysis and 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1% NP-40, and visualization were done in PartekGS software. Functional assignment of a protease inhibitor mixture (Complete; Roche, Mannheim, Germany). the genes was performed using the “Functional Annotation Tools” in IL-10 and IFN-g ELISA were performed using reagents and protocols DAVID bioinformatics resources (http://david.abcc.ncifcrf.gov) following provided by the manufacturer (eBioscience and BioLegend, respectively). recommended protocols. Additional analysis was performed using In- Samples were plated in duplicate. genuity Pathways Analysis (Ingenuity Systems, Redwood City, CA).

Quantification of demyelination qRT-PCR Blinded quantification of demyelination was performed using Luxol fast GFP2 and GFP+ CD8 T cells were sorted as described earlier, centrifuged, blue-stained sections as previously described (19). and resuspended in TRIzol Reagent (Invitrogen/Life Technologies, Carls- bad, CA). RNA was extracted and reverse-transcribed using Superscript II Affymetrix microarray (Invitrogen). Relative cytokine transcript quantities were determined by qRT-PCR using SYBR Green (SA Biosciences, Frederick, MD). Primers 2 GFP+CD8+ and GFP CD8+ T cells harvested from infected Vert-X mouse used for qRT-PCR were as follows: IL-10: forward, 59-GCGTCGTGA- brains were sorted at day 7 p.i., as described earlier. RNA was purified TTAGCGATGATG-39; reverse, 59-CTCGAGCAAGTCTTTCAGTCC-39; using RNeasy columns (Qiagen) according to manufacturer instructions. TNF: forward, 59-TCAGCCGATTTGCTATCTCA-39; reverse, 59-CGGA-

RNA samples were verified for purity spectroscopically, and the quality of CTCCGCAAAGTCTAAG-39; TLR2: forward, 59-TTTCGTTCATCTC- Downloaded from the intact RNA was assessed using an Agilent 2100 Bioanalyzer. RNA for TGGAGCA-39; reverse, 59-GAGTCCGGAGGGAATAGAGG-39; TLR3: the microarray was processed using a NuGEN WT-Ovation Pico RNA forward, 59-ACCTTTGTCTTCTGCACGAACCT-39; reverse, 59-AGTTC- Amplification System together with a NuGEN WT-Ovation Exon Module. TTCACTTCGCAACGCA-39; TLR4: forward, 59-TCAGAACTTGAGT- Samples were hybridized and loaded onto Affymetrix GeneChip Mouse GGCTGCA-39; reverse, 59-GAGGCCAATTTTGTCTCCAC-39; TLR7: GENE 1.0 ST arrays. Arrays were scanned with an Affymetrix Model 7G forward, 59-CAGCCATAACCAGCTGACAA-39; reverse, 59-TTGCAAA- upgraded scanner, and data were collected using GeneChip Operating GAAAGCGATTGTG-39; CCL2: forward, 59-AGCACCAGCCAACTCT- Software. Complete microarray data have been deposited at the Gene CACT-39; reverse, 59-TCATTGGGATCATCTTGCTG-39; IL-1a: forward, Expression Omnibus under accession number GSE25846 (http://www. 59-CACAACTGTTCGTGAGCGCT-39; reverse, 59-TTGGTGTTTCTGG- http://www.jimmunol.org/ ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25846). CAACTCCT-39; IL-1b: forward, 59-ACTGTTTCTAATGCCTTCCC -39; reverse, 59-ATGGTTTCTTGTGACCCTGA-39; STAT-4: forward, 59- Analysis of microarray data CCTACTGGCAGAGAGTCTTTTCC-39; reverse, 59-GGTTGTAGATCA- GGAAGGTAGC-3 9; perforin: forward, 59-TTGGCCCATTTGGTGGT- Data from the Affymetrix Mouse Exon 1.0 ST arrays were first quantile AAG-39; reverse, 59-AGTCTCCCCACAGATGTTCTGC-39; prosaposin: normalized and median polished using Robust Multichip Average back- forward, 59-ACTTTCCCACCACCTGTAGC-39; reverse, 59-TAACTGCA- + 2 ground correction with log2 adjusted values. GFP and GFP samples were CAGGCTGTCTCC-39; HEXA: forward, 59-GTGCTCATGAAGTCGT- obtained from the same mice, allowing for a paired t test. Partek batch AGGTGC-39; reverse, 59-CAGGATGTGAAGGAGGTCATTG-39; hypo- correction was used to remove variation caused by the hybridization batch. xanthine-guanine phosphoribosyltransferase: forward, 59-GCGTCGTGAT- by guest on September 28, 2021

FIGURE 2. IL-10 is produced by T cells in the brain. Vert-X mice were infected with J2.2-V-1 and sacrificed at stated time points. A, CNS-derived lymphocytes were examined for IL-10 production at day 7 p.i., as detected by GFP staining. B, Total numbers of IL-10+ T cells recovered in the brain at different days p.i. C, Relative contribution of GFP+ cells from various cell types (calculated by multiplying frequency of GFP+ by frequency of cell type) in the brain at stated time points. D, IL-10 protein levels in brain homogenates were determined by ELISA at the stated time points. E, MFI of IL-10 production from various cell types at day 7 p.i. in the brain. ***p , 0.001. Data are from three separate experiments at each time point with 12 mice/group. The Journal of Immunology 3645

TAGCGATCATC-39; reverse, 59-CTCGAGCAAGTCTTTCAGTCC-39. Results Data were analyzed as previously described (18). IL-10 is produced at the site of infection, the brain, by virus-specific T cells Cytotoxicity assay

2 + Previous work demonstrated that IL-10 protein is expressed in the GFP and GFP CD8 T cells were sorted as described earlier. EL4 target CNS of J2.2-V-1–infected mice (5, 21, 22). To identify the cellular cells were peptide pulsed with 1 mM S510 peptide at 37˚C for 1 h and washed. Cytolytic activity was determined using a Cytotox 96 non- sources of IL-10 during the acute and recovery stages of viral radioactive (Promega, Madison, WI) according to the manufacturer’s encephalitis, we infected IL-10–GFP reporter (Vert-X) mice (5, instructions. Cytotoxicity assays were performed in triplicate. 13, 17) with J2.2-V-1 and monitored brain, draining cervical lymph nodes (CLN), spleen, and peripheral blood for GFP+ cells Complement depletion from days 0–42 p.i. These time points were chosen because virus JHMV-immune splenocytes were prepared from B6 or IL-102/2 mice is cleared by day 14 p.i. and inflammation is mostly resolved by immunized with 3 3 105 PFU wild-type JHMV i.p. 7 d before adoptive day 42 p.i. (14, 23). We observed the highest frequency of IL-10– transfer. Donor splenocytes were depleted of CD4 T lymphocytes, as producing cells at day 7 p.i., coincident with the onset of the previously described (20). Greater than 99% depletion was achieved, as adaptive immune response (Fig. 1A,1B). Uninfected mice (Fig. assessed by FACS. 1B) and mice at days 2 and 4 p.i. (data not shown) had very low + Statistics levels of GFP cells. At all time points, the highest frequency of GFP+ cells was found in the brain, and not in the CLN, spleen, or Two-tailed unpaired Student t tests were used to analyze differences in peripheral blood (Fig. 1B–D, peripheral blood not shown). In mean values between groups. Fisher’s exact tests were used to analyze ∼ Downloaded from differences in survival. One-way ANOVAs were used to analyze differ- addition, T cells accounted for 90% of IL-10 production (Fig. ences in multiple groups. All results are expressed as means 6 SEs of the 2A). Thus, IL-10 is produced by T cells specifically at the site of means. Differences of p values ,0.05 were considered significant. infection during peak inflammation. http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. IL-10 is produced by virus-specific effector T cells. Vert-X (tetramer staining) or B6 (intracellular cytokine staining [ICS]) mice were infected with 500 PFU J2.2-V-1 and sacrificed at day 7 p.i. For ICS, CNS-derived B6 lymphocytes were prepared and stimulated with the indicated peptides and stained for IL-10, IFN-g, and TNF expression. A, Upper and lower rows are gated on CD4 and CD8 T cells, respectively, and show tetramer/GFP staining (left panels) or cytokine expression after peptide stimulation (right panels). B, Frequency of M133-specific CD4 T cells that coproduce IL-10 and IFN-g or TNF is shown. C, Frequency of S510-specific CD8 T cells that coproduce IL-10 and IFN-g or TNF is shown. D, Cells were costained for IL-10, Foxp3, T-bet, IL-4, and/or IL-17, and examined by flow cytometry. FACS plots are gated on CD4 T cells. Data are from four separate experiments with a total of 12 mice analyzed. 3646 HYPERACTIVATED CYTOTOXIC CD8 T CELLS EXPRESS IL-10

During the peak of J2.2-V-1 infection, more CD8 T cells than infected B6 mice (Fig. 3A) (23, 25). In addition, we examined CD4 T cells infiltrate the CNS (24, 25). Therefore, although IFN-g and TNF expression by IL-10+CD8 T cells, using intra- a greater fraction of CD4 than CD8 T cells expressed IL-10, cellular cytokine staining after stimulation with peptides corres- ∼50% of the IL-10–producing cells were CD8+ (Fig. 2B,2C). ponding to S510 and the subdominant epitope, S598. In these The frequency of IL-10+CD8 T cells diminished from 40% at day assays, we identified IL-10+ cells by intracellular cytokine staining 7 p.i. to ,5% at day 21 p.i., whereas the frequency of IL-10+CD4 because GFP diffused from cells under conditions necessary for T cells declined more slowly over the course of infection (Fig. IFN-g and TNF detection. Nearly all of the virus epitope-specific 2C). Consistent with the numbers of IL-10–producing T cells, IL- IL-10+CD8 T cells expressed IFN-g and 60–70% also produced 10 protein levels peaked in the brain at day 7 p.i. and diminished TNF (Fig. 3C). Furthermore, cells recognizing the two peptides thereafter (Fig. 2D). Of note, at all time points, the mean fluo- accounted for almost all of the IL-10 produced by CD8 T cells rescence intensity (MFI) of GFP expression was greater in CD4 (Fig. 3A,3C). Interestingly, IFN-g and TNF were expressed by compared with CD8 T cells, demonstrating greater IL-10 pro- virus-specific IL-10+CD8 T cells at greater levels per cell than IL- duction on a per cell basis (Fig. 2E) (17). 102CD8 T cells (MFI for S510-specific CD8 T cells: IFN-g+ Because IL-10 production is generally indicative of a T cell with cells: 418 6 8 versus 217 6 4, p = 0.0001; TNF+ cells: 305 6 23 suppressor function (26), we next determined whether the IL-10+ versus 169 6 11; p = 0.0007 for IL-10+ and IL-102 cells, re- CD8 T cells were as potent effectors as IL-102CD8 T cells. Using spectively) consistent with a more highly activated phenotype. MHC class I/peptide tetramer staining, we observed that approx- Most IL-10+CD4 T cells in the brains of J2.2-V-1–infected mice imately half of the GFP+ cells were specific for S510, the were effector Th1 T cells, with the majority coproducing IFN-g immunodominant CD8 T cell epitope recognized in J2.2-V-1– and TNF when stimulated by immunodominant (M133) and Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. CD8 IL-10 production requires recent antigenic stimulation and ERK and p38 MAPK activation for maximal IL-10 production. A, Brain lymphocytes from J2.2-V-1 Vert-X mice were harvested at day 7 p.i. and stained for CD69, KLRG-1, and CD127 (IL-7R). Shown are representative FACS plots gated on CD8 T cells. B, Brain lymphocytes were obtained from three J2.2-V-1–infected Vert-X mice day 7 p.i. GFP+(IL-10+) and GFP+(IL-102) CD8 T cells were sorted and cultured with varying concentrations of S510 peptide-pulsed irradiated splenocytes for 48 h. IL-10 and IFN-g production by GFP+ CD8 T cells were detected by ELISA. C, CNS-derived lymphocytes were examined directly ex vivo for pERK expression. Histogram of pERK in IL-10+ CD8 or IL-102CD8 T cells and bar graph displaying MFI for pERK are shown. D and E, Brain lymphocytes were cultured in vitro as in B with stated concentrations of MEK1/2 inhibitor PD184161 (ERK1/2) (D) or p38 inhibitor PD169316 (E) and analyzed for IL-10 production. F, Levels of IL-10 in culture supernatants after 48 h were quantified by ELISA. Samples contained GFP+ or GFP2 CD8 T cells and irradiated splenocytes in the presence or absence of 1 mM S510 peptide-pulsed irradiated splenocytes. Control wells included irradiated splenocytes but no added CD8 T cells. *p , 0.05, **p , 0.01, ***p , 0.001. Data are representative of three to six separate experiments. The Journal of Immunology 3647 subdominant (S358) peptides (Fig. 3A,3B). Of note, a lower Strong signaling through the TCR activates the MAPK pathway, percentage of M133-specific CD4 T cells were identified using consisting of ERKs (ERK1/2), JUN N-terminal kinases, and p38 (4, tetramer than by intracellular cytokine expression, consistent with 31). Because IL-10 production was dependent on stimulation with low affinity of TCR to MHC class II/peptide binding (27). Ap- high Ag concentrations, we next examined whether the MAPK proximately 10–15% of IL-10+CD4 T cells expressed Foxp3, pathway was activated under conditions of high Ag stimulation. expressed by regulatory T cells, whereas 85–90% expressed T-bet, As shown in Fig. 4C, the level of p-ERK was greater in IL-10+ consistent with a Th1 phenotype (Fig. 3D). J2.2-V-1 infection CD8 T cells than in IL-102CD8 T cells when cells were stained induces a strong Th1 cytokine response (21, 28, 29). Only a very directly ex vivo for p-ERK1 and p-ERK2 (p44/42). low number of cells in the J2.2-V-1–infected brain expressed IL-4 To demonstrate a functional role for ERK, we stimulated GFP+ or IL-17; consequently, few, if any, of the IL-10+ cells also CD8 T cells with irradiated splenocytes pulsed with S510 peptide expressed either of these cytokines (Fig. 3D). in the presence of varying concentrations of the ERK inhibitor PD184161 for 48 h in an in vitro assay. PD184161 is a potent, IL-10 production by CD8 T cells requires strong Ag stimulation selective inhibitor of MEK1/2, the upstream kinases of ERK1/2 and signaling through the MAPK pathway (32). As seen in Fig. 4D, selective inhibition of ERK phosphor- High doses of stimulatory Ag are required for IL-10 compared with ylation decreased IL-10 production by ∼50%. Another MAPK, IFN-g production by CD4 T cells (6). CD8 T cells produced IL-10 p38, was also involved in IL-10 expression because stimulation of at the site of infection and production was maximal at the peak of GFP+CD8 T cells with S510 peptide in the presence of a selective the inflammatory response (Fig. 1). Furthermore, a greater fre- p38 inhibitor (PD169316) (33, 34) decreased IL-10 production by + quency of IL-10 CD8 T cells expressed CD69, a marker of recent 75% (Fig. 4E). Decreased amounts of IL-10 did not reflect a loss Downloaded from 2 antigenic stimulation, compared with IL-10 CD8 T cells (Fig. of cell viability because only low levels of apoptosis (∼10%), as 4A). In addition, CD127 expression was slightly higher and assessed by Annexin V and 7-AAD staining, were detected in all + KLRG-1 expression modestly lower on IL-10 CD8 T cells, sug- cultures (data not shown). Therefore, inhibition of either ERK1/2 gestive of a memory progenitor phenotype (Fig. 4A) (30). This and p38 MAPK pathways decreased IL-10 expression by CD8 raised the possibility that IL-10 production in CD8 T cells was T cells.

also dependent on strong Ag stimulation. To assess this possibility, http://www.jimmunol.org/ we examined IL-10 production by stimulating GFP+CD8 T cells IL-10 production by CD8 T cells is transient with irradiated splenocytes pulsed with varying S510 peptide Unexpectedly, when we stimulated GFP2CD8 cells for 48 h with doses directly ex vivo for 48 h. Indeed, ∼100-fold more peptide high concentrations of peptide S510, we detected IL-10 in the was required to produce 50% maximal levels of IL-10 compared cultures, suggesting that the sorted GFP2CD8 T cells had con- with IFN-g (Fig. 4B). verted to IL-10–producing cells after high antigenic stimulation by guest on September 28, 2021

FIGURE 5. The production of IL-10 by effector CD8 and CD4 T cells is transient in vivo. Brain lymphocytes were obtained from three J2.2-V-1–infected Vert-X mice day 7 p.i. GFP+(IL-10+)Thy1.2 and GFP2(IL-102)Thy1.2 CD4 and CD8 T cells were sorted and adoptively transferred (AT) i.v. into a J2.2-V- 1–infected Thy1.1 mouse at day 1 p.i. Six days later, mice were sacrificed. A, Histograms show GFP expression before and after sorting, and 6 d after AT. B and C, Frequency of AT CD4 (B) or CD8 (C) Thy1.2 lymphocytes in the brain, CLN, and spleen. D, Frequency of AT Thy1.2+ cells that were GFP+ in the brain. White bars denote mice originally receiving GFP2Thy1.2 lymphocytes; black bars are mice that originally received GFP+Thy1.2 lymphocytes. Data are representative of five separate experiments with a total of 10 mice analyzed. 3648 HYPERACTIVATED CYTOTOXIC CD8 T CELLS EXPRESS IL-10

IL-10 produced by CD8 T cells is suppressive Because J2.2-V-1–specific IL-10+CD8 T cells coproduced IFN-g and TNF, we assessed whether the IL-10 produced by these cells was immunosuppressive or, alternatively, immunostimulatory (35, 36) in an in vitro proliferation assay. Using a transwell assay system, we placed GFP+CD8 T cells in the upper compartment with peptide-stimulated irradiated splenocytes. CFSE-labeled naive lymphocytes were incubated in the lower compartment of an anti-CD3–coated plate. As expected, naive CD4 and CD8 T lymphocytes proliferated in the presence of anti-CD3 stimula- tion as demonstrated by CFSE dilution (Fig. 6). Proliferation was inhibited if GFP+CD8 T cells were present in the culture. In- FIGURE 6. IL-10 expressed by cytotoxic CD8 T cells is suppressive hibition was mediated by IL-10, because the presence of a block- directly ex vivo. CNS-derived GFP+(IL-10+) CD8 T cells were sorted ing IL-10 Ab largely reversed the inhibitory effect. and cultured with S510-peptide pulsed-irradiated splenocytes in an upper transwell compartment, whereas CFSE-labeled naive lymphocytes were CD8 T cells that produce IL-10 express a transcriptional placed in the bottom compartment and stimulated with anti-CD3 Ab for hyperactivated signature 48 h. Cells were cultured with blocking anti–IL-10 Ab or with an isotype + control. Histograms show CFSE dilution of lower compartment CD4 and IL-10 CD8 T cells were most abundant during the peak of in- Downloaded from CD8 lymphocytes. The upper compartment contained GFP+CD8 (A)orno flammation (Fig. 1B) and coproduced inflammatory cytokines CD8 T cells (B). Data are representative of three separate experiments. (Fig. 3C), and IL-10 expression was dependent on MAPK acti- vation and high Ag load (Fig. 4). These results, coupled with data (Fig. 4F). Consistent with this result, some of the cells that were showing that these cells were immunosuppressive (Fig. 6), raised initially GFP2 expressed GFP after in vitro culture (data not the possibility that the IL-10+CD8 T cells were more highly ac- 2

shown). IL-10 expression did not occur in the absence of peptide tivated than IL-10 CD8 T cells. To evaluate this possibility, we http://www.jimmunol.org/ 2 S510 or in cultures without sorted CD8 T cells (Fig. 4F). compared patterns of global transcription in IL-10+ and IL-10 To investigate whether this phenotypic plasticity also occurred in CD8 T cells by microarray analyses. the infected animal, we transferred Thy1.2 GFP+ T cells or Thy1.2 For this purpose, CNS-derived CD8 T lymphocytes were pooled GFP2 T cells into J2.2-V-1–infected Thy1.1 mice. Mice were from three mice and sorted for recent IL-10 production as detected sacrificed 6 d after transfer, and Thy1.2 CD8 and CD4 T cells by GFP expression to high purity (.99%) at day 7 after J2.2-V-1 were examined for GFP expression. Mice were analyzed at day 7 infection. Genome-wide microarray studies were performed on p.i. to allow sufficient time for proliferation of transferred cells. mRNA purified from three pools of lymphocytes. We performed The highest frequency of transferred cells, whether initially GFP+ statistics on the data and found 384 genes sta- 2 + or GFP , was detected in the infected brain compared with the tistically significantly increased and 25 genes decreased in IL-10 by guest on September 28, 2021 2 CLN or spleen (Fig. 5B, 5C). In addition, transferred GFP+ and cells compared with IL-10 cells (Supplemental Table I). Using GFP2 T cells were detected in the brain at similar frequencies, DAVID and Ingenuity Pathways Analysis to evaluate the gene sets suggesting that IL-10+ cells were not more prone to cell death. identified, we detected significant changes in several pathways of Remarkably, IL-10 production was transient in both CD8 and CD4 immune activation and regulation (Table I, Supplemental Table I). T cells, so that some cells that were initially IL-10+ (GFP+) be- Notably, there was increased expression of genes encoding came GFP2 and some of the IL-102 (GFP2) T cells became GFP+ cytokines (Il1a, Il1b, Il10, Il18, Tnf), chemokines (Ccl2, Ccl3, (Fig. 5A,5D). Of note, T cells that were originally IL-10+ or IL- Ccl6, Ccl7, Ccl8, Ccl9, Ccl12, Cxcl2, Cxcl4 Cxcl6, Cxcl9), im- 102 appeared to proliferate equally, even though IL-10 inhibited mune sensor molecules (Tlr1, Tlr2, Tlr3, Tlr4, Tlr6, Tlr8, Tlr13, proliferation in vitro. This equivalent proliferative ability may Fcer1g, Fcgr1), and lysosomes/endocytosis (Abca1 [ATPeV], reflect IL-10 plasticity, because similar frequencies of cells were Gla, Hexa/b, Lamp1/2 [CD107a/b], Acp2, Prf1, Psap, Lgmn, ctsb/ IL-102 after 6 d in mice. c/h/l/z [cathepsin B/C/H/L/Z]), suggesting that cells expressing

Table I. Increased gene expression in inflammatory pathways in IL-101CD8 T cells compared with IL-102CD8 T cells

No. of %of KEGG Pathway Genes Input p Value Genes Upregulated Genes Downregulated Lysosome 21 5.9 2.15E-10 CTSZ LIPA, PSAP, HEXA, GUSB, LGMN, HEXB, ACP2, CD63, ASAH1, GNS, LAMP1, SLC11A1, CTSL, LAMP2, CD68, GLA, ATP6V0A1, CTSC, CTSB, CTSH Cytokine–cytokine 26 7.3 4.69E-08 CCL3, CCL2, TNF, IL18, CCR1, CXCL2, CCL9, CXCL9, CCL8, CCR9, IL18R1, interaction PF4, IL10, CCL7, CCL6, IL1B, CSF3R, IL13RA1, IFNGR2, IL1A, IL18RAP, XCL1 CSF1R, TGFBR1, HGF, OSM, CCL12, INHBA, CXCL16, TNFSF12-TNFSF13 TLR signaling pathway 16 4.5 1.85E-07 CCL3, TNF, PIK3CB, LY96, TLR1, CXCL9, TLR2, TLR3, TLR4, TLR7, TLR8, FOS, JUN, IL1B, CD14, SPP1 signaling 17 4.8 9.72E-05 CCL3, CCL2, LYN, PIK3CB, HCK, CCR1, CXCL2, CXCL9, pathway CCL9, CCL8, PF4, CCL7, CCL6, CCL12, GNGT2, CXCL16, PAK1 Ag processing and 8 2.3 0.018 CTSL, LGMN, H2-EB1, IFI30, H2-AB1, CTSB, H2-DMA, CD74 presentation Genome-wide mRNA expression in IL-101CD8 T cells was analyzed using microarrays and compared with IL-102CD8 T cells. Genes with significant changes were placed into the indicated biological pathways using the KEGG pathway tool in DAVID bioinformatics resources. Genes upregulated or downregulated refers to genes increased or decreased in IL-101CD8 compared with IL-102CD8 T cells. The Journal of Immunology 3649

IL-10 also exhibited a highly activated proinflammatory tran- were all increased in epitope S510-specific IL-10+CD8 compared scriptional profile. with IL-102CD8 T cells (Fig. 7C), confirming our microarray To validate these results, we analyzed a subset of these differ- data. Most importantly, IL-10+CD8 T cells were more cytotoxic entially regulated genes by qRT-PCR and for protein expression. than IL-102CD8 T cells when examined directly ex vivo for the As shown in Fig. 7A, IL-1a, IL-1b, IL-10, CCL2, TLR2, ability to lyse peptide S510-coated EL4 target cells (Fig. 7D). TLR3, TLR4, prosaposin, hexosaminidase A, and perforin mRNA were increased and STAT-4 mRNA was decreased in IL-10+CD8 IL-10 is protective in J2.2-V-1 demyelinating disease compared with IL-102CD8 T cells, confirming our microarray Demyelination in J2.2-V-1–infected mice is largely immuno- analysis. As shown in Fig. 4A, some IL-102CD8 T cells were pathological, with myelin damage occurring during virus clear- CD69+, indicating recent Ag exposure. To confirm that these cells ance (20). The observation that IL-10+CD8 T cells were highly were not hyperactivated, we sorted CD69+IL-102, CD69+IL-10+, activated and cytolytic raised the question whether the IL-10 and CD692IL-102 CD8 T cells, and compared expression levels expressed by these cells would be able to diminish immunopath- of a subset of genes shown in Fig. 7A. CD69+IL-10+CD8 T cells ological changes in the J2.2-V-1–infected CNS. had increased IL-10, IL-1a, CCL2, TLR3, and perforin, and de- First, we confirmed a role for IL-10 in J2.2-V-1–infected mice by creased STAT-4 mRNA levels compared with CD69+IL-102CD8 monitoring survival, weight, and demyelination in age- and sex- T cells (Fig. 7B). In addition, we investigated protein expression in matched IL-10+/+ and IL-102/2 B6 mice infected with 1500 PFU IL-10+ cells by surface staining for MHC class II and the high- J2.2-V-1. Infected IL-102/2 mice died at a greater rate than IL- affinity IgE receptor (FcεR1), and by intracellular staining for 10+/+ mice and had increased weight loss (Fig. 8A; days 7–12 p.i.; 2 2 LAMP1/2 (CD107a/b) and granzyme B after stimulation with p # 0.05). We detected more rapid virus clearance in IL-10 / Downloaded from peptide S510. LAMP1/2, granzyme B, MHC class II, and FcεR1 mice, showing that the mice had increased ability to control the http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. qRT-PCR and protein analyses show increased expression of inflammatory pathways in IL-10+CD8 T cells compared with IL-102CD8 T cells. A, qRT-PCR was performed on IL-10+CD8 and IL-102CD8 T cell mRNA purified from CNS-derived lymphocytes. Genes were selected for analysis based on the microarray results. B, qRT-PCR was performed on CD692IL-102CD8, CD69+IL-102CD8, and CD69+IL-10+CD8 T cell mRNA purified from CNS-derived lymphocytes. CD69+IL-10+ and CD69+IL-102 CD8 T cell samples were analyzed for statistically significant differences. C, CNS-derived lymphocytes from a J2.2-V-1–infected B6 mouse were cultured with S510 peptide for 6 h and then costained with IL-10, CD107a/b (LAMP1/ 2), and/or granzyme B. Cells from infected Vert-X mice were stained for MHC class II and FcεR1 expression. All FACS plots are gated on CD8 T cells. D, CNS-derived IL-10+ and IL-102 CD8 lymphocytes were analyzed for ex vivo cytolytic function using S510-peptide–coated EL4 cells. Effector/target ratios were determined based on percentage of tetramer S510+ effector cells. *p , 0.05, **p , 0.01, ***p , 0.001. Data are representative of two or three separate experiments with a total of 8–12 mice analyzed. 3650 HYPERACTIVATED CYTOTOXIC CD8 T CELLS EXPRESS IL-10 infection. Diminished IL-10 expression is often associated with 102/2 CD8 T cells (Fig. 8C,8D; days 9–12 p.i.; p # 0.05). Thus, increased numbers of inflammatory cells present at sites of in- IL-10 expressed by CD8 T cells was protective in the context of flammation (2). Consistent with these results, we found more in- infection with J2.2-V-1. nate immune effector cells (macrophages, neutrophils, and NK cells) at day 4 p.i. and increased virus-specific CD4 and CD8 Discussion T cells at day 8 p.i. (Fig. 9). Thus, increased cellular infiltration in In this study, we demonstrate that virus-specific CD8 T cells that the CNS of IL-102/2 mice enhanced viral clearance, but at the are most cytotoxic and hyperactivated in mice with acute en- cost of diminishing survival. To assess the role of IL-10 in de- cephalitis express IL-10 during the height of the inflammatory myelination, we examined infected spinal cords for myelin de- response. One interpretation of these results is that IL-10 functions struction at day 21 p.i., the peak of demyelination (37). For these in a self-suppressing or localized manner by tempering the im- experiments, we infected mice with 500 PFU J2.2-V-1 to enhance mune response induced by these highly activated CD8 T cells. In survival (Fig. 8B). Demyelination, as assessed by Luxol fast blue this scenario, CD8 T cells would self-regulate when viral Ag staining, was significantly increased in IL-102/2 mice when com- concentration (Fig. 4B) and inflammatory response reach a cer- pared with B6 mice (Fig. 8B). tain level, to minimize immunopathological disease (e.g., de- To directly examine the individual contribution of IL-10 myelination) (Fig. 8). Because only the most activated virus- expressed by CD8 T cells, we adoptively transferred virus- specific cells express IL-10, immunosuppression may not be immune undepleted or CD8 T cell-enriched splenocytes from generalized, but localized to sites where bystander damage would IL-10+/+ or IL-102/2 mice to IL-102/2 mice 1 d p.i. with 1500 be maximal. IL-10 expression by CD8 T cells was also detected

PFU J2.2-V-1. Similar numbers of epitope S510-specific T cells during the peak of inflammation in mice infected with influenza A Downloaded from were present in the transferred IL-10+/+ and IL-102/2 splenocytes virus or SV5 (10, 13). Although there is not agreement in all (data not shown). Although there was no difference in survival studies (12), inflammation is increased and clinical disease wor- between mice receiving CD8 T cell-enriched splenocytes from B6 sens in the absence of IL-10 in both infections. Furthermore, we or IL-102/2 mice, there was diminished weight loss and de- show that IL-10 expression solely by CD8 T cells was protective myelination in mice receiving IL-10–replete compared with IL- in an adoptive transfer system, even though expression was highly http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. IL-10 produced from CD8 T cells is protective in chronic encephalomyelitis. A, IL-10+/+ and IL-102/2 B6 mice were infected with 1500 PFU J2.2-V-1 and monitored for survival, weight, and virus titers. Dashed line depicts the limit of virus detection. Differences in survival reached statistical significance (p = 0.02). B, IL-10+/+ and IL-102/2 B6 mice were infected with 500 PFU J2.2-V-1 and monitored for survival (left) and spinal cord de- myelination at day 21 p.i. Each symbol represents a different mouse. C and D, IL-102/2 B6 mice were infected with 1500 PFU J2.2-V-1 and received no cells or undepleted or CD8 T cell-enriched splenocytes from virus-immune IL-10+/+ or IL-102/2 B6 mice. Mice were monitored for survival and weight loss (C), and spinal cord demyelination (D). *p , 0.05, ***p , 0.001. Data are representative of three separate experiments, with a total of 15 mice analyzed. The Journal of Immunology 3651

ERK1/2 signaling but not p38 is required for IL-10 production in all CD4 Th subsets (6, 31). Therefore, IL-10 expression is de- pendent on MAPK pathways in all tested cells, but exact re- quirements are cell specific. Although we did not examine the role of JNK of the MAPK pathway in IL-10 expression, its sig- naling may also be needed for maximal IL-10 production as IL- 10+ cells showed increased jun expression as assessed by gene expression array (Supplemental Table I). Our results showed that IL-10+CD8 T cells were more cytolytic than IL-102CD8 T cells (Fig. 7D). Consistent with these data, genes involved in cytolytic function were increased in IL-10+CD8 compared with IL-102CD8 T cells in gene expression analyses (Fig. 7, Table I). Other upregulated genes in IL-10+CD8 T cells included several that are involved in the innate immune response and are most commonly expressed by and macro- phages. Some of these proteins, such as the chemokines CCL2, CCL8, CCL12, and CXCL4 ( factor 4) and cytokines IL- 1a, IL-1b, and IL-18 are predicted to enhance the inflammatory

response. However, further work will be required to understand Downloaded from why some genes, such as TLRs, C-type lectins, and FcRs, are increased in expression in IL-10+CD8 T cells. Equally interesting is diminished expression of STAT-4 in IL- 10+CD8 T cells; STAT-4 is required for maximal IL-12–induced IL-10 expression in CD4 T cells (6). CD8 T cell expression of IL-

10 is likely to be STAT-4 and thus, IL-12, independent because we http://www.jimmunol.org/ + FIGURE 9. IL-102/2 mice have increased brain cell infiltrates com- found the same frequency of IL-10 CD8 T cells in the brains of IL- 2/2 pared with IL-10+/+ mice. IL-10+/+ and IL-102/2 mice were infected with 12Rb2 and wild type B6 mice (data not shown). Recently, the 1500 PFU J2.2-V-1. CNS-derived leukocytes were stained as described in transcription factors c-Maf and Ahr (aryl hydrocarbon receptor) Materials and Methods and examined by flow cytometry. Mice were were demonstrated to be essential for IL-10 expression by Tr1 CD4 2 2 sacrificed at day 4 (A) or 8 p.i. (B). White bars represent IL-10 / mice; T cells and c-Maf for IL-10 production in macrophages (43, 44). +/+ black bars represent IL-10 B6 mice. *p , 0.05, **p , 0.01. Data are The expression of these transcription factors was not increased in representative of two separate experiments. IL-10+CD8 T cells, possibly because they may be upregulated only during the early induction phase. Alternatively, different pathways elevated for only a short period (Figs. 1, 8). In addition, expression of IL-10 induction may exist in CD4 and CD8 T cells. by guest on September 28, 2021 of IL-10 by these highly activated CD8 cells was dynamic, as In summary, IL-10 is produced by the most highly activated, shown by the high rate of interconversion of IL-102 and IL-10+ cytotoxic CD8 T cells in the infected CNS at the peak of in- populations in vitro and in vivo after adoptive transfer. This flammation and is protective against morbidity. This study is con- plasticity may reflect recent exposure to virus Ag because the IL- sistent with the notion that cytotoxic CD8 T cells self-regulate 10+CD8 T cells preferentially expressed CD69 (Fig. 4A). Variable themselves by producing IL-10, and thus that enhanced activa- IL-10 expression suggests that exposure to Ag is transitory, pos- tion does not invariably result in the development of cells with sibly reflecting focal distribution of virus Ag in the brain (38). greater potential for immunopathological disease. Manipulation of Furthermore, virus is cleared from the brain in a rostral to caudal IL-10 expression by CD8 T cells might afford another approach to manner (39, 40), with clearance of inflammatory cells lagging diminishing tissue destruction in animals or humans with acute behind that virus (S. Perlman, unpublished observations); cells encephalitis. remaining at sites of virus clearance would have diminished viral Ag exposure resulting in downregulation of IL-10 production. The Acknowledgments high activation state of these cells was not a precursor to cell We thank Drs. John Harty, Hai-Hui Xue, and Jon Houtman for stimulating + death, because IL-10 cells could be detected for at least 6 d after discussion and critical review of the manuscript. We thank Dr. Tom Bair adoptive transfer. (University of Iowa DNA facility) for help with gene expression analysis Until recently, IL-10 has been considered to be most important in and the National Institutes of Health Tetramer Core Facility (Atlanta, chronic infections and autoimmune disease. In animals persistently GA) for providing tetramers. infected with lymphocytic choriomeningitis virus or Leishmania or HCV-infected humans, IL-10 produced by CD4 T cells has been Disclosures implicated in pathogen persistence (7–9, 41). In these infections, The authors have no financial conflicts of interest. IL-10 was produced by CD4 and not CD8 T cells; this is con- sistent with our results because IL-10 expression by CD8 T cells References rapidly diminishes after day 7 p.i. and is nearly at background 1. Sellon, R. K., S. Tonkonogy, M. Schultz, L. A. Dieleman, W. Grenther, levels by 21 d p.i. in the brain, whereas CD4 T cells continue to E. Balish, D. M. Rennick, and R. B. Sartor. 1998. Resident enteric bacteria are express IL-10 for at least 42 d p.i. (Fig. 1). necessary for development of spontaneous colitis and activation To our knowledge, this is the first report to demonstrate that in -10-deficient mice. Infect. Immun. 66: 5224–5231. 2. Couper, K. N., D. G. Blount, and E. M. Riley. 2008. IL-10: the master regulator IL-10 expression in CD8 T cells is dependent on high Ag stimula- of immunity to infection. J. Immunol. 180: 5771–5777. tion and signaling through the ERK1/2 and p38 MAPK pathways 3. Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O’Garra. 2001. Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19: 683–765. (Fig. 4C–E). p38, but not ERK1/2 signaling, was found to be 4. Saraiva, M., and A. O’Garra. 2010. The regulation of IL-10 production by im- important in IL-10 production in monocytes (42). Conversely, mune cells. Nat. Rev. Immunol. 10: 170–181. 3652 HYPERACTIVATED CYTOTOXIC CD8 T CELLS EXPRESS IL-10

5. Perona-Wright, G., K. Mohrs, F. M. Szaba, L. W. Kummer, R. Madan, 25. Haring, J. S., L. L. Pewe, and S. Perlman. 2001. High-magnitude, virus-specific C. L. Karp, L. L. Johnson, S. T. Smiley, and M. Mohrs. 2009. Systemic but not CD4 T-cell response in the central nervous system of coronavirus-infected mice. local infections elicit immunosuppressive IL-10 production by natural killer J. Virol. 75: 3043–3047. cells. Cell Host Microbe 6: 503–512. 26. O’Garra, A., and K. M. Murphy. 2009. From IL-10 to IL-12: how pathogens and 6. Saraiva, M., J. R. Christensen, M. Veldhoen, T. L. Murphy, K. M. Murphy, and their products stimulate APCs to induce T(H)1 development. Nat. Immunol. 10: A. O’Garra. 2009. Interleukin-10 production by Th1 cells requires interleukin- 929–932. 12-induced STAT4 transcription factor and ERK MAP kinase activation by high 27. Landais, E., P. A. Romagnoli, A. L. Corper, J. Shires, J. D. Altman, I. A. Wilson, antigen dose. Immunity 31: 209–219. K. C. Garcia, and L. Teyton. 2009. New design of MHC class II tetramers to 7. Brooks, D. G., M. J. Trifilo, K. H. Edelmann, L. Teyton, D. B. McGavern, and accommodate fundamental principles of antigen presentation. J. Immunol. 183: M. B. Oldstone. 2006. Interleukin-10 determines viral clearance or persistence 7949–7957. in vivo. Nat. Med. 12: 1301–1309. 28. Held, K. S., W. G. Glass, Y. I. Orlovsky, K. A. Shamberger, T. D. Petley, 8. Ejrnaes, M., C. M. Filippi, M. M. Martinic, E. M. Ling, L. M. Togher, S. Crotty, P. J. Branigan, J. M. Carton, H. S. Beck, M. R. Cunningham, J. M. Benson, and and M. G. von Herrath. 2006. Resolution of a chronic viral infection after T. E. Lane. 2008. Generation of a protective T-cell response following corona- interleukin-10 receptor blockade. J. Exp. Med. 203: 2461–2472. virus infection of the central nervous system is not dependent on IL-12/23 sig- 9. Belkaid, Y., K. F. Hoffmann, S. Mendez, S. Kamhawi, M. C. Udey, T. A. Wynn, naling. Viral Immunol. 21: 173–188. and D. L. Sacks. 2001. The role of interleukin (IL)-10 in the persistence of 29. Anghelina, D., L. Pewe, and S. Perlman. 2006. Pathogenic role for virus-specific Leishmania major in the skin after healing and the therapeutic potential of anti- CD4 T cells in mice with coronavirus-induced acute encephalitis. Am. J. Pathol. IL-10 receptor antibody for sterile cure. J. Exp. Med. 194: 1497–1506. 169: 209–222. 10. Palmer, E. M., B. C. Holbrook, S. Arimilli, G. D. Parks, and M. A. Alexander- 30. Kaech, S. M., and E. J. Wherry. 2007. Heterogeneity and cell-fate decisions in Miller. 2010. IFNgamma-producing, virus-specific CD8+ effector cells acquire effector and memory CD8+ T cell differentiation during viral infection. Immu- the ability to produce IL-10 as a result of entry into the infected lung environ- nity 27: 393–405. ment. Virology 404: 225–230. 31. Dong, C., R. J. Davis, and R. A. Flavell. 2002. MAP kinases in the immune 11. Spender, L. C., T. Hussell, and P. J. Openshaw. 1998. Abundant IFN-gamma response. Annu. Rev. Immunol. 20: 55–72. production by local T cells in respiratory syncytial virus-induced eosinophilic 32. Klein, P. J., C. M. Schmidt, C. A. Wiesenauer, J. N. Choi, E. A. Gage, M. T. Yip- lung disease. J. Gen. Virol. 79: 1751–1758. Schneider, E. A. Wiebke, Y. Wang, C. Omer, and J. S. Sebolt-Leopold. 2006. The 12. McKinstry, K. K., T. M. Strutt, A. Buck, J. D. Curtis, J. P. Dibble, G. Huston, effects of a novel MEK inhibitor PD184161 on MEK-ERK signaling and growth Downloaded from M. Tighe, H. Hamada, S. Sell, R. W. Dutton, and S. L. Swain. 2009. IL-10 in human liver cancer. Neoplasia 8: 1–8. deficiency unleashes an influenza-specific Th17 response and enhances sur- 33. Gallagher, T. F., G. L. Seibel, S. Kassis, J. T. Laydon, M. J. Blumenthal, vival against high-dose challenge. J. Immunol. 182: 7353–7363. J. C. Lee, D. Lee, J. C. Boehm, S. M. Fier-Thompson, J. W. Abt, et al. 1997. 13. Sun, J., R. Madan, C. L. Karp, and T. J. Braciale. 2009. Effector T cells control Regulation of stress-induced cytokine production by pyridinylimidazoles; in- lung inflammation during acute influenza virus infection by producing IL-10. hibition of CSBP kinase. Bioorg. Med. Chem. 5: 49–64. Nat. Med. 15: 277–284. 34. Davies, S. P., H. Reddy, M. Caivano, and P. Cohen. 2000. Specificity and 14. Bergmann, C. C., T. E. Lane, and S. A. Stohlman. 2006. Coronavirus infection of mechanism of action of some commonly used protein kinase inhibitors. Bio-

the central nervous system: host-virus stand-off. Nat. Rev. Microbiol. 4: 121–132. chem. J. 351: 95–105. http://www.jimmunol.org/ 15. Stohlman, S. A., C. C. Bergmann, and S. Perlman. 1998. Mouse hepatitis virus. 35. Groux, H., M. Bigler, J. E. de Vries, and M. G. Roncarolo. 1998. Inhibitory and In Persistent viral infections. R. Ahmed, and I. Chen, eds. John Wiley & Sons, stimulatory effects of IL-10 on human CD8+ T cells. J. Immunol. 160: 3188–3193. Ltd., New York. p. 537–557. 36. Santin, A. D., P. L. Hermonat, A. Ravaggi, S. Bellone, S. Pecorelli, J. J. Roman, 16. Zhao, J., J. Zhao, and S. Perlman. 2009. De novo recruitment of antigen- G. P. Parham, and M. J. Cannon. 2000. Interleukin-10 increases Th1 cytokine experienced and naive T cells contributes to the long-term maintenance of an- production and cytotoxic potential in human papillomavirus-specific CD8(+) tiviral T cell populations in the persistently infected central nervous system. J. cytotoxic T lymphocytes. J. Virol. 74: 4729–4737. Immunol. 183: 5163–5170. 37. Wang, F. I., D. R. Hinton, W. Gilmore, M. D. Trousdale, and J. O. Fleming. 17. Madan, R., F. Demircik, S. Surianarayanan, J. L. Allen, S. Divanovic, 1992. Sequential infection of glial cells by the murine hepatitis virus JHM strain A. Trompette, N. Yogev, Y. Gu, M. Khodoun, D. Hildeman, et al. 2009. Non- (MHV-4) leads to a characteristic distribution of demyelination. Lab. Invest. 66: redundant roles for -derived IL-10 in immune counter-regulation. J. 744–754. Immunol. 183: 2312–2320. 38. Barnett, E. M., M. D. Cassell, and S. Perlman. 1993. Two neurotropic viruses,

18. Trandem, K., D. Anghelina, J. Zhao, and S. Perlman. 2010. Regulatory T cells herpes simplex virus type 1 and mouse hepatitis virus, spread along different by guest on September 28, 2021 inhibit T cell proliferation and decrease demyelination in mice chronically neural pathways from the main olfactory bulb. Neuroscience 57: 1007–1025. infected with a coronavirus. J. Immunol. 184: 4391–4400. 39. Perlman, S., G. Jacobsen, and A. Afifi. 1989. Spread of a neurotropic murine 19. Xue, S., N. Sun, N. Van Rooijen, and S. Perlman. 1999. Depletion of blood- coronavirus into the CNS via the trigeminal and olfactory nerves. Virology 170: borne macrophages does not reduce demyelination in mice infected with a neu- 556–560. rotropic coronavirus. J. Virol. 73: 6327–6334. 40. Perlman, S., G. Evans, and A. Afifi. 1990. Effect of olfactory bulb ablation on spread 20. Wu, G. F., A. A. Dandekar, L. Pewe, and S. Perlman. 2000. CD4 and CD8 T cells of a neurotropic coronavirus into the mouse brain. J. Exp. Med. 172: 1127–1132. have redundant but not identical roles in virus-induced demyelination. J. 41. Brady, M. T., A. J. MacDonald, A. G. Rowan, and K. H. Mills. 2003. Hepatitis C Immunol. 165: 2278–2286. virus non-structural protein 4 suppresses Th1 responses by stimulating IL-10 21. Kapil, P., R. Atkinson, C. Ramakrishna, D. J. Cua, C. C. Bergmann, and production from monocytes. Eur. J. Immunol. 33: 3448–3457. S. A. Stohlman. 2009. Interleukin-12 (IL-12), but not IL-23, deficiency ameli- 42. Foey, A. D., S. L. Parry, L. M. Williams, M. Feldmann, B. M. Foxwell, and orates viral encephalitis without affecting viral control. J. Virol. 83: 5978–5986. F. M. Brennan. 1998. Regulation of IL-10 synthesis by endogenous 22. Lin, M. T., D. R. Hinton, B. Parra, S. A. Stohlman, and R. C. van der Veen. 1998. IL-1 and TNF-alpha: role of the p38 and p42/44 mitogen-activated protein The role of IL-10 in mouse hepatitis virus-induced demyelinating encephalo- kinases. J. Immunol. 160: 920–928. myelitis. Virology 245: 270–280. 43. Apetoh, L., F. J. Quintana, C. Pot, N. Joller, S. Xiao, D. Kumar, E. J. Burns, 23. Templeton, S. P., and S. Perlman. 2007. Pathogenesis of acute and chronic D. H. Sherr, H. L. Weiner, and V. K. Kuchroo. 2010. The aryl hydrocarbon central nervous system infection with variants of mouse hepatitis virus, strain receptor interacts with c-Maf to promote the differentiation of type 1 regulatory JHM. Immunol. Res. 39: 160–172. T cells induced by IL-27. Nat. Immunol. 11: 854–861. 24. Williamson, J. S., K. C. Sykes, and S. A. Stohlman. 1991. Characterization of 44. Cao, S., J. Liu, L. Song, and X. Ma. 2005. The protooncogene c-Maf is an es- brain-infiltrating mononuclear cells during infection with mouse hepatitis virus sential transcription factor for IL-10 gene expression in macrophages. J. strain JHM. J. Neuroimmunol. 32: 199–207. Immunol. 174: 3484–3492. Table S1: Functional annotation of all genes with significant changes in IL-10+/IL-10- CD8 T cells.a Gene Description Fold change Gene Description Fold change Gene Description Fold change Gene Description Fold change Lysosome Endocytosis ctnd. Transcription Miscellaneous ctnd. 5430435G22Rik RIKEN cDNA 5430435G22 3.7808 Fcgr3 Fcr IgG, low affinity III 4.1067 Ang angiogenin 3.6841 Hpgds 3.4124 Acp2 acid phosphatase 2 3.5973 Hck hemopoietic cell kinase 2.5686 Atf3 activating transcription 3 4.7633 Hsd17b11 2.236 Asah1 N-acylsphingosine hydrolase 3.2073 Lrp1 LDL receptor-related 1 2.8889 Atp6v0a1 ATPase, H+ transporting 2.4885 Htr2b serotonin receptor 2B 2.2575 Cd63 CD63 antigen 4.3914 Mertk c-mer tyrosine kinase 4.3677 Bhlhe41 basic HLH family, e41 2.1417 Idh1 2.3648 Cd68 CD68 antigen 3.3325 Mrc1 , C type 1 3.8822 Cebpa C/EBP, alpha 2.0812 Ier3 immediate early response 3 3.1134 Cd74 CD74 antigen 2.7124 Msr1 macro scavenger receptor 1 5.9684 D14Ertd668e predicted gene 6907 3.4285 Ifitm2 induced protein 2 2.2106 Clcn7 chloride channel 7 2.1862 Ophn1 oligophrenin 1 2.664 Ell2 elongation RNA polymerase 2.6003 Igsf6 Ig superfamily, member 6 4.1667 Ctsb cathepsin B 2.9977 Rin2 like Ras and Rab interactor 2.3081 Fos cellular oncogene c-fos 2.1303 Iigp1 2.1155 Ctsc cathepsin C 2.4768 Sirpa signal-regulatory protein 4.0744 Ifi204 interferon activated 204 2.3503 Il13ra1 IL-13 receptor, alpha 1 2.7082 Ctsh cathepsin H 4.4672 Slc11a1 solute carrier family 11 2.0286 Ifi205 interferon activated 205 4.6612 Il18bp 2.9358 Ctsl cathepsin L 3.5933 Jun Jun oncogene 2.193 Il1rn 2.8158 Ctsz cathepsin Z 3.4067 Antigen processing and presentation Mafb v-maf 3.6512 Inhba 2.234 Gla galactosidase, alpha 2.707 Cd74 CD74 antigen 2.7124 Myc myelocytomatosis -2.1365 Irg1 3.4628 Gns glucosamine sulfatase 2.3769 Fcer1g Fcr IgE, high affinity I 3.6578 Nr1h3 nuclear receptor family 1 2.0887 Itga9 integrin alpha 9 2.0426 Gusb glucuronidase, beta 2.2846 Fcgr1 Fcr IgG, high affinity I 4.1133 Rora RAR-related orphan receptor -2.1249 Itgb5 integrin beta 5 3.5269 H2-DMa histocompatibility 2, class II 2.6441 Fcgr2b Fcr IgG, low affinity IIb 3.1371 Sfpi1 SFFV proviral integration 1 2.377 Kmo kynurenine 3-hydroxylase -2.1013 Hexa hexosaminidase A 3.8264 Fcgr3 Fcr IgG, low affinity III 4.1067 Stat4 STAT 4 -2.0636 Lair1 leukocyte-associated Ig-like 2.6281 Hexb hexosaminidase B 3.1652 H2-Ab1 histocompatibility 2 Ab1 2.1787 Tceal8 transcription elongation A 3.4798 Lap3 2.5402 Hpse heparanase 2.4165 H2-DMa histocompatibility 2 DMa 2.6441 Tcf7 transcription factor 7 -2.5021 LOC100044195 2.291 Ifi30 inducible 3.701 H2-Eb1 histocompatibility 2 Eb1 2.0444 Tcf7l2 transcription factor 7-like 2.5021 Lpcat2 lpc acyltransferase 2 3.0531 Lamp1 CD107a 2.053 Ifi30 interferon gamma inducible 3.701 Tcfec transcription factor EC 2.3481 Ly86 4.0742 Lamp2 CD107b 2.031 Slc11a1 solute carrier family 11 2.0286 Zeb2 zinc finger E-box binding 2.6381 Ly96 2.9262 Lgmn legumain 5.7916 Unc93b1 unc-93 homolog B1 2.1214 Lyz2 4.094 Lipa lysosomal acid lipase A 2.161 Miscellaneous Map4k3 2.411 Naaa N-acylethanolamine amidase 2.0925 Carbohydrate binding 1810011H11Rik 2.8926 Marcks myristoylated PK C 4.283 Prf1 perforin 1 (pore forming) 2.2924 Ang angiogenin 3.6841 3110043O21Rik 2.1218 Mgst1 4.7365 Psap prosaposin 2.6672 Apoe apolipoprotein E 4.0103 4930506M07Rik 2.049 Mmp14 matrix metallopeptidase 14 3.2217 Slc11a1 solute carrier family 11 2.0286 App amyloid beta (A4) 4.6465 5033414K04Rik 2.1406 Mmp8 matrix metallopeptidase 8 2.2174 Slc15a3 solute carrier family 15 3.8469 Ccl7 chemokine ligand 7 3.7248 5730408K05Rik -2.3711 Mospd2 motile sperm 2 2.0467 Ccl8 chemokine ligand 8 11.851 5830405N20Rik -2.4306 Mpeg1 expressed 2.81 Cytokines/Chemokines Cd72 CD72 antigen 3.0112 9030625A04Rik 4.3524 Ms4a14 membrane-spanning 4A, 14 4.7621 Ccl12 chemokine ligand 12 6.219 Cd93 CD93 antigen 4.1967 A430084P05RikRIKEN cDNA A430084P05 2.6867 Ms4a4a membrane-spanning 4A, 4A 2.3562 Ccl2 chemokine ligand 2 4.8683 Cfh complement factor h 2.3239 AA467197 3.3507 Ms4a6c membrane-spanning 4A, 6C 2.2827 Ccl3 chemokine ligand 3 2.5405 Chi3l3 Chitinase 3-like protein 3 4.4019 Abcc3 ATP-binding cassette 3.0573 Ms4a6d membrane-spanning 4A, 6D 3.0385 Ccl6 chemokine ligand 6 3.0471 Clec12a C-type lectin 12, member a 3.8098 Abhd12 abhydrolase domain 12 2.1439 Ms4a7 membrane-spanning 4A, 7 5.9117 Ccl7 chemokine ligand 7 3.7248 Clec2i C-type lectin 2, member i -2.039 Acsl1 acyl-CoA synthetase 1 2.1924 Myo1e 2.5164 Ccl8 chemokine ligand 8 11.851 Clec4a1 C-type lectin 4, member a1 3.1678 Adam8 ADAM 8 2.2451 Myof myoferlin 2.9554 Ccl9 chemokine ligand 9 2.3226 Clec4a3 C-type lectin 4, member a3 3.3332 Adap2 3.0993 Naip2 2.1531 Cxcl16 chemokine ligand 16 3.6658 Clec4d C-type lectin 4, member d 4.8543 AF251705 cDNA sequence AF251705 3.2759 Naip5 2.2774 Cxcl2 chemokine ligand 2 4.5322 Clec4e C-type lectin 4, member e 5.1436 AI607873 -2.202 Ncf2 2.4868 Cxcl4 6.9198 Clec5a C-type lectin 5, member a 3.8504 Aif1 2.7865 Neb 2.2864 Cxcl9 chemokine ligand 9 2.8768 Clec7a C-type lectin 7, member a 3.4043 Akr1e1 2.1923 Olfml3 3.6812 Grn granulin 4.0943 Gm156 predicted gene 156 2.0032 Alox5ap arachidonate 5-lipoxygenase 3.3395 OTTMUSG00000003606 2.4529 Il10 4.1716 Gpnmb glycoprotein nmb 6.448 Anpep alanyl aminopeptidase 5.1823 OTTMUSG00000010657 2.2201 Il18 2.0876 Itgam 2.5778 Anxa3 2.9554 P2rx4 purinergic receptor P2X 3.6285 Il1a interleukin 1 alpha 12.327 Klra2 KLR, subfamily A2 3.0084 Anxa4 2.449 P2ry13 purinergic receptor P2Y 2.7421 Il1b interleukin 1 beta 2.5012 Mrc1 mannose receptor, C1 3.8822 Aoah 3.5438 P2ry6 pyrimidinergic receptor P2Y 3.569 Osm 2.9809 Pf4 platelet factor 4 3.8822 Apobec1 2.965 Pak1 2.4002 Spp1 secreted phosphoprotein 1 6.1037 Thbs1 thrombospondin 1 2.7039 Apoc2 3.1742 Pde7b 2.8089 Tnf 2.949 Arg1 4.5679 Pdlim1 -2.9881 Tnfsf12-tnfsf13 TNF (ligand) superfamily 3.12 Cell activation Art2b ADP-ribosyltransferase 2b -2.9037 Pdpn podoplanin 2.9623 Tnfrsf26 TNF receptor superfamily -2.2732 Cd74 CD74 antigen 2.7124 Asph aspartate-beta-hydroxylase 2.065 Pfkfb3 2.446 Entpd1 ectonucleoside triphosphate 2.3276 Atg4c 2.1473 Pgcp 2.8031 Chemotaxis Fcer1g Fcr IgE, high affinity I 3.6578 Atp1a3 ATPase, Na+/K+ transport 2.3085 Pilra paired Ig-like type 2 2.4615 C3ar1 complement 3a receptor 1 4.4441 Fcgr2b Fcr IgG, low affinity IIb 3.1371 B230118H07Rik 2.1657 Pira11 2.3773 C5ar1 complement 5a receptor 1 4.2916 Fcgr3 Fcr IgG, low affinity III 4.1067 BC013712 2.5347 Pira2 3.3949 Ccl12 chemokine ligand 12 6.219 H2-DMa histocompatibility 2 DMa 2.6441 BC028528 3.6254 Pla2g4a 3.6816 Ccl2 chemokine ligand 2 4.8683 Itgam integrin alpha M 2.5778 Bhlhe41 2.1417 Pla2g7 8.2232 Ccl3 chemokine ligand 3 2.5405 Klrg1 KLR subfamily G, member 1 -2.583 Bst1 BM stromal cell antigen 1 3.1017 Plau 4.3431 Ccl6 chemokine ligand 6 3.0471 Lat2 linker for activation T cells 2.0681 Capg 2.7266 Plaur plasminogen activator 2.2183 Ccl7 chemokine ligand 7 3.7248 Lilrb3 Leukocyte Ig-like receptor 2.0025 Car13 2.069 Plbd1 3.6087 Ccl8 chemokine ligand 8 11.851 Lst1 leukocyte specific 1 3.08 Ccdc88a 2.0565 Pld4 phospholipase D, member 4 2.4215 Ccl9 chemokine ligand 9 2.3226 Pf4 platelet factor 4 6.9198 Ccdc90b coiled-coil domain 90B 2.0494 Plekho1 2.7092 Ccr1 1 3.7591 Pik3cb PI 3-kinase 2.2783 Cd180 CD180 antigen 4.8975 Plin2 3.6091 Ccr9 chemokine receptor 9 -2.3116 Sfpi1 SFFV proviral integration 1 2.377 Cd244 CD244 NK receptor 2B4 2.5188 Plk2 4.846 Ccrl2 chemokine receptor-like 2 3.6478 Skap2 src family associated 2 2.3592 Cd300ld RIKEN cDNA 4732429D16 2.3275 Pltp 2.0099 Csf1r CSF 1 receptor 4.9352 Slc11a1 solute carrier family 11 2.0286 Cd300lf CD300 antigen like family 2.8884 Plxdc2 plexin domain containing 2 2.6536 Csf3r CFS 3 receptor 2.068 Tcf7 transcription factor 7 -3.149 Cd83 CD83 antigen 2.5598 Plxnb2 plexin B2 2.1025 Cxcl16 chemokine ligand 16 3.6658 Timp1 TIMP 1 2.157 Cdh17 cadherin 17 2.7802 Pros1 2.701 Cxcl2 chemokine ligand 2 4.5322 Tlr1 toll-like receptor 1 2.7198 Ch25h cholesterol 25-hydroxylase 5.2625 Ptgs1 2.0742 Cxcl4 platelet factor 4 6.9198 Tlr4 toll-like receptor 4 4.7295 Ckb 2.2399 Ptgs2 4.2389 Cysltr1 cysteinyl leukotriene receptor 2.1644 Tnf tumor necrosis factor 2.949 Cmklr1 chemokine-like receptor 1 2.5904 Pygl 2.121 Fcer1g Fcr IgE, high affinity I 3.6578 Tpd52 tumor protein D52 2.5819 Cpd carboxypeptidase D 2.961 Rab32 2.6574 Fcgr3 Fcr IgG, low affinity III 4.1067 Cpne2 2.9996 Rap2a 2.0755 Il1b interleukin 1 beta 2.5012 Protein kinase cascade Creg1 3.1394 Rasgef1b 3.6757 Il18r1 interleukin 18 receptor 1 -3.0498 Btk bruton .8073 Cst3 2.4204 Rcan1 2.255 Il18rap IL-18 receptor accessory -2.3976 Fgd4 FYVE, RhoGEF and PH 2.9488 Cstb 2.493 Renbp 2.7836 Itgam integrin alpha M 2.5778 Il1b interleukin 1 beta 2.5012 Cttnbp2nl 2.2023 Rgl1 3.1802 Nrp2 2 2.4811 Lst1 leukocyte transcript 1 3.08 Cybb cytochrome b-245 2.7702 Rhob ras homolog, member B 3.5866 Ptafr platelet-activating receptor 2.5969 Lyn Yamaguchi sarcoma homolog 3.7161 Cyfip1 3.0054 Rnf130 ring finger protein 130 2.9467 Xcl1 chemokine ligand 1 -3.4851 Osm oncostatin M 2.9809 Cyp4f18 cytochrome P450, family 4 3.2973 Rnf149 ring finger protein 149 2.0402 Pde2a phosphodiesterase 2A -2.007 Dapl1 -2.202 Rrbp1 ribosome binding protein 1 2.0244 Immune effector process Psap prosaposin 2.6672 Dhrs3 dehydrogenase/reductase 3.0279 Saa3 6.8762 C1qa complement 1, qa 5.7723 Tgm2 transglutaminase 2 3.3365 Dmxl2 2.4351 Samd3 -2.2978 C1qb complement 1, qb 3.2695 Timp2 TIMP 2 3.2779 Dnase1l1 2.0742 Sash1 2.5605 C1qc complement 1, qc 4.9011 Tlr3 toll-like receptor 3 2.442 Dock4 2.7166 Sat1 2.059 C3 complement component 3 3.3697 Tlr4 toll-like receptor 4 4.7295 Dusp1 2.2206 Scamp1 secretory carrier membrane 2.1876 Cd14 CD14 antigen 4.9916 Tnf tumor necrosis factor 2.949 E430029J22Rik -2.1465 Scoc 2.0731 Cd226 CD226 antigen -2.1869 Zeb2 zinc finger E-box 2 2.6381 Ecm1 3.2114 Scpep1 2.6037 Cd74 CD74 antigen 2.7124 Emb embigin -2.1912 Sepp1 3.3042 Cfb complement factor B 4.3704 MAPKKK cascade Emr1 4.9337 Sgk1 2.8206 Cfh complement factor h 2.3239 Cd74 CD74 antigen 2.7124 Epb4.1l2 2.0211 Sh2d1b1 2.0056 Clec7a C-type lectin family 7a 3.4043 Cd81 CD81 antigen 3.5648 Ets2 2.1737 Sidt1 SID1 transmembrane family -2.4457 Fcer1g Fcr IgE, high affinity I 3.6578 Fgd4 FYVE, RhoGEF and PH 2.9488 Etv5 2.4717 Sirpb1 3.4628 Fcgr1 Fcr IgG, high affinity I 4.1133 Hgf hepatocyte 4.6048 F10 2.197 Slamf8 SLAM family member 8 3.6816 Fcgr2b Fcr IgG, low affinity IIb 3.1371 Slc11a1 solute carrier family 11 2.0286 F11r 3.7776 Slc31a2 solute carrier family 31, 2 2.0362 Fcgr3 Fcr IgG, low affinity III 4.1067 Spred1 sprouty protein with EVH-1 2.0754 Fabp5 5.7232 Slc35f5 solute carrier family 35, F5 2.2913 Fcgr4 Fcr IgG, low affinity IV 4.5212 Tnf tumor necrosis factor 2.949 Fabp7 3.8449 Slc7a11 solute carrier family 7, 11 2.0536 Fgl2 -like 2 2.4251 Fam46c 2.2221 Slc7a7 solute carrier family 7, 7 2.8311 H2-DMa histocompatibility 2 DMa 2.6441 Apoptosis Regulation Fcrls 3.2895 Slc7a8 solute carrier family 7, 8 3.1846 Ifngr2 interferon-g receptor 2 2.7759 Apoe apolipoprotein E 4.0103 Fmnl2 3.0445 Slfn4 3.1249 Lat2 linker for activation T cells 2.0681 Bnip3 E1B interacting protein 3 3.02 Fnip2 3.2497 S1pr1 S-I-P receptor 1 -2.9075 Lilrb3 Leukocyte Ig-like receptor 2.0025 Cd74 CD74 antigen 2.7124 Ftl1 2.5884 Snx24 2.457 Lyn Yamaguchi sarcoma homolog 3.7161 Cdkn1a cyclin-d kinase inhibitor 1A 2.265 Ftl2 2.4351 Sparc 4.7802 Nlrp3 NLR family, pyrin domain 3 2.5673 Fcer1g Fcr IgE, high affinity I 3.6578 Fzd7 frizzled homolog 7 2.0218 Speer1-ps1 2.4255 Slc11a1 solute carrier family 11 2.0286 Fcgr1 Fcr IgG, high affinity I 4.1133 Gab2 2.0459 Sqrdl 2.9826 Tlr2 toll-like receptor 2 3.3915 Fcgr3 Fcr IgG, low affinity III 4.1067 Gatm 5.9161 Stom stomatin 2.3976 Tlr4 toll-like receptor 4 4.7295 Hgf hepatocyte growth factor 4.6048 Gcnt1 glucosaminyl transferase 1 2.1116 Tbc1d9 2.3442 Tlr7 toll-like receptor 7 2.8304 Hmox1 heme oxygenase 1 3.2747 Gcnt2 glucosaminyl transferase 2 2.3185 Tcn2 2.2791 Tlr8 toll-like receptor 8 2.0831 Ifi204 interferon activated gene 2.3503 Gda 2.8918 Tctex1d2 2.4845 Tlr13 toll-like receptor 13 6.001 Il10 interleukin 10 4.1716 Gm10134 3.5893 Tec 2.0561 Tnfsf12-tnfsf13 TNF (ligand) superfamily 3.12 Il18 interleukin 18 2.0876 Gm11428 6.5574 Tgfbi 4.0142 Unc93b1 unc-93 homolog B1 2.1214 Lst1 leukocyte specific 1 3.08 Gm4951 3.8104 Thap6 2.4765 Niacr1 niacin receptor 1 2.0481 Gm5150 3.6608 Tifa 2.1157 Endocytosis Nlrp3 NLR family, pyrin domain 3 2.5673 Gm5431 3.1213 Tmbim1 BAX inhibitor motif 1 2.1354 Abca1 ATP-binding cassette 3.984 Niacr3 nuclear protein 1 2.3255 Gm6377 5.1929 Tmem104 transmembrane protein 104 2.1354 App amyloid beta precursor 4.6465 Spp1 secreted phosphoprotein 1 6.1037 Gm6455 2.8655 Tmem106a transmembrane protein 106A 2.0284 Cd36 CD36 antigen 4.3224 Tgfbr1 TNF beta receptor I 2.1184 Gngt2 2.2543 Tmem119 transmembrane protein 119 3.273 Clec7a C-type lectin domain 7a 3.4043 Tgm2 transglutaminase 2, 3.3365 Gpr137b-ps 2.6627 Tmem77 2.5353 Dab2 disabled homolog 2 4.599 Timp1 TIMP 1 2.157 Gpr141 G protein-coupled receptor 2.1791 Trem1 triggering receptor 2.2478 Fcer1g Fcr IgE, high affinity I 3.6578 Tnf tumor necrosis factor 2.949 Gpr183 G protein-coupled receptor -2.8038 Trf 2.3152 Fcgr1 Fcr IgG, high affinity I 4.1133 Gpr84 G protein-coupled receptor 2.4729 Tyrobp TYRO tyrosine kinase BP 2.1272 Fcgr2b Fcr IgG, low affinity IIb 3.1371 Hist1h2bc 2.0657 Vwa5a 2.513 Hk3 2.1609 Xlr 3.071 aGenome-wide mRNA expression in IL-10+CD8 T cells was analyzed via microarray and compared to IL-10-CD8 T cells. Biological pathways analysis of certain genes with mRNA changes was created using DAVID bioinformatics tool. Note that some genes were placed into more than one classification.