CD28 Deficiency Enhances Type I IFN Production by Murine Plasmacytoid Dendritic Cells

This information is current as Monica Macal, Miguel A. Tam, Charles Hesser, Jeremy Di of September 29, 2021. Domizio, Psylvia Leger, Michel Gilliet and Elina I. Zuniga J Immunol 2016; 196:1900-1909; Prepublished online 15 January 2016; doi: 10.4049/jimmunol.1501658

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD28 Deficiency Enhances Type I IFN Production by Murine Plasmacytoid Dendritic Cells

Monica Macal,* Miguel A. Tam,* Charles Hesser,* Jeremy Di Domizio,† Psylvia Leger,* Michel Gilliet,† and Elina I. Zuniga*

Type I IFNs (IFN-I) are key innate mediators that create a profound antiviral state and orchestrate the activation of almost all immune cells. Plasmacytoid dendritic cells (pDCs) are the most powerful IFN-I–producing cells and play important roles during viral infections, cancer, and autoimmune diseases. By comparing expression profiles of murine pDCs and conventional DCs, we found that CD28, a prototypic T cell stimulatory receptor, was highly expressed in pDCs. Strikingly, CD28 acted as a negative regulator of pDC IFN-I production upon TLR stimulation but did not affect pDC survival or maturation. Importantly, cell- intrinsic CD28 expression restrained pDC (and systemic) IFN-I production during in vivo RNA and DNA viral infections, limiting antiviral responses and enhancing viral growth early after exposure. Finally, CD28 also downregulated IFN-I response upon skin Downloaded from injury. Our study identified a new pDC regulatory mechanism by which the same CD28 molecule that promotes stimulation in most cells that express it is co-opted to negatively regulate pDC IFN-I production and limit innate responses. The Journal of Immunology, 2016, 196: 1900–1909.

ype I IFNs (IFN-I) play a crucial role in orchestrating the . Not only do pDCs produce up to 1000 times more IFN-

immune response to multiple disease settings, including I than other cell types but they also synthesize a broader range of http://www.jimmunol.org/ T viral infections, cancers, tissue injury, and autoimmune IFN-I isoforms (3). pDCs express endosomal TLR7 and TLR9, disease (1). IFN-I is a pleiotropic family found among which recognize ssRNA and unmethylated CpG-containing motifs mammalian species that includes several IFN-a and one IFN-b (from microbial or self-origin), respectively (4). Engagement of isoforms that signal through a common ubiquitously expressed TLR7 or TLR9 in pDCs leads to production of IFN-I (both IFN-a receptor (IFNab-R), promoting both autocrine and paracrine ac- and IFN-b isoforms) as well as proinflammatory cytokines and tivation and leading to phosphorylation of STAT 1 and 2. The upregulation of costimulatory molecules such as CD80, CD86, result of these interactions is a positive feedback loop that drives and MHC class II (MHC-II) (5–10). further IFN-I production as well as the induction of hundreds of As such, pDCs play an important role during several in vivo viral IFN-I–stimulated (ISGs) (2). These ISGs act in concert to infections such as those caused by murine CMV (MCMV) (11, 12), by guest on September 29, 2021 create a potent antiviral state and orchestrate the activation of respiratory syncytial virus (13, 14), and mouse hepatitis virus (15), almost all innate and adaptive immune cells. Although almost all among others (15–17). Furthermore, persistent viruses such as cell types can produce IFN-I, plasmacytoid dendritic cells (pDCs) HIV and hepatitis C virus induce substantial IFN-I production are highly specialized to rapidly secrete copious amounts of these upon incubation with pDCs (17, 18), and similar effects are ob- served early after in vivo infection with persistent strains of lymphocytic choriomeningitis virus (LCMV WE or clone 13; *Division of Biological Sciences, University of California San Diego, La Jolla, CA Cl13) (19, 20). However, pDC IFN-I production becomes 92093; and †Service de Dermatologie et ve´ne´re´ologie, Centre Hospitalier Universi- exhausted during later stages of chronic viral infection, an event taire Vaudois, University Hospital of Lausanne, Lausanne CH-1011, Switzerland accompanied by enhanced susceptibility to opportunistic patho- ORCIDs: 0000-0001-8094-7090 (M.A.T.); 0000-0002-6690-3283 (C.H.); 0000- gens (18, 21–23). Similarly, pDC IFN-I production is also atten- 0002-6281-3918 (J.D.D.). uated in tumor microenvironments, correlating with cancer Received for publication July 24, 2015. Accepted for publication December 9, 2015. progression (24). In contrast, uncontrolled IFN-I production by This work was supported by the Lupus Research Institute, the American Cancer pDCs is associated with autoimmune diseases such as psoriasis Society, and National Institute of Health Grant A1081923 (to E.I.Z.). E.I.Z. is a Leukemia and Lymphoma Society scholar. M.M. was supported by National Insti- (25), type I diabetes (26), and experimental autoimmune en- tutes of Health Supplemental Award A1081923. M.A.T. was supported by a post- cephalomyelitis (27). In particular, in systemic lupus erythe- doctoral fellowship from the Swedish Research Foundation. matosus patients, pDCs accumulate in target tissues and exhibit The microarray data presented in this article have been submitted to the National sustained IFN-I production, and pDCs were shown to be critical Center for Biotechnology Information’s Gene Expression Omnibus (https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE75834) under accession number for promoting systemic lupus erythematosus pathogenesis (28– GSE75834. 30). Finally, pDC IFN-I production also promotes innate defenses Address correspondence and reprint requests to Dr. Elina I. Zuniga, Division of following tissue injury, playing a critical role in regulating cuta- Biological Sciences, University of California San Diego, 9500 Gilman Drive 0322, neous wound healing (31). Taken together, these studies demon- La Jolla, CA 92093-0322. E-mail address: [email protected] strate the importance of fine-tuning the magnitude of pDC IFN-I The online version of this article contains supplemental material. response and highlight the significant implications of pDC IFN-I Abbreviations used in this article: BM, bone marrow; cDC, conventional DC; Cl13, clone 13; dko, double knockout; Flt3L, Flt3 ligand; ISG, IFN-I–stimulated gene; regulation for numerous human illnesses. LCMV, lymphocytic choriomeningitis virus; MCMV, murine CMV; MHC-II, MHC In the current study, we compared the gene expression profiles of class II; pDC, plasmacytoid dendritic cell; PI, propidium iodide; p.i., postinfection; pDCs and conventional DCs (cDCs) to gain insight on putative qPCR, quantitative PCR; WT, wild-type. pDC IFN-I regulators. Unexpectedly, we found that CD28, a cell Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 surface stimulatory receptor constitutively expressed in T cells www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501658 The Journal of Immunology 1901

(32), was highly and selectively expressed in pDCs but not cDCs. Cytokine detection Remarkably, CD28 expression negatively regulated pDC IFN-I Total IFN-I bioactivity was measured by luciferase bioassay with reference production in response to in vitro TLR stimulation and in vivo to a recombinant mouse IFN-b standard (Research Diagnostics, Concord, viral infections or tissue injury. Moreover, bone marrow (BM) MA) using a L-929 cell line transfected with an IFN-sensitive luciferase as chimeras revealed a cell-intrinsic effect of CD28 expression in described previously (37). TNF-a and IL-6 were measured by ELISA (eBio- suppressing pDC functions. Thus, our study identified a novel role science). Supernatant cytokine levels were measured 15 h poststimulation. for the prototypic T cell stimulatory molecule CD28 as a negative Flow cytometry regulator of pDC function both in vitro and in vivo. Considering The following Abs were used to stain murine BM or spleen cells: anti–CD3- that CD28 is fundamental for T cell priming (32), our work raises PerCP-Cy5.5 (145-2C11; BD Biosciences), Thy 1.2-PE (53-2.1), CD19-PE the possibility that CD28 may be part of a previously unrecog- (eBio1D3), NK 1.1-PE (PK136), CD11c-PE or allophycocyanin (N418), nized molecular pathway that inhibits innate responses while CD11b-PE-Cy7 (M1/70), B220-allophycocyanin eFluor 780 or efluor 450 promoting adaptive immunity. (RA3-6B2), PDCA-1-FITC (eBio927), CD45.2-allophycocyanin-Cy7 (104), CD45.1-PerCy5.5 (A20), MHC-II (I-A/I-E) efluor 450 (M5/114.15.2), CD28-allophycocyanin (E18) and mouse IgG2b-allophycocyanin isotype Materials and Methods control for anti–CD28-allophycocyanin (eBioscience), and CD86 PE Cy7 Mice and viruses or PE (IT2.2; BioLegend). PI or Ghost dye (Tonbo Biosciences, San Diego, CA) was used to exclude dead cells and to measure cell viability where C57BL/6 (wild-type [WT]), C57BL/6 CD45.1+, CD80/86 double knockout indicated. Cells were acquired with an LSRII flow cytometer (BD Bio- (dko), and CD28ko mice were purchased from The Jackson Laboratory sciences). Data were analyzed with FlowJo software. (Bar Harbor, ME). Mice (6–12 wk old) were infected i.p. with 1 3 104 PFU MCMV Smith or 2 3 106 LCMV Cl13 i.v. Viruses were propagated Generation of mixed WT:CD28ko BM chimeras Downloaded from and quantified as described previously (33, 34). WT CD45.1+ C57BL/6 recipient mice were sublethally irradiated with + Mechanical injury of mouse skin and analysis of dermal cells 1000 rad and reconstituted with a 50:50 mix of BM cells from CD45.1 WT mice and CD45.2+ CD28ko mice. 10 3 106 BM cells were transferred Mice backs were shaved and depilated (Veet; Reckitt Benckiser, Slough, i.v. into the irradiated recipient mice, which were treated with antibiotics U.K.) immediately before injury. Mechanical injury was then applied by (8 mg/ml trimethoprim and 40 mg/ml sulfamethoxazole supplied in tape stripping, using 20 strokes of transparent tape (3M; Scotch) across the drinking water) for 3 wk to prevent infection and allow immune recon- back. Injured skin was excised, digested with 1 mg/ml dispase (Sigma- stitution. Reconstitution was analyzed 8 wk after BM transfer, and the ratio http://www.jimmunol.org/ Aldrich, St. Louis, MO), 200 U/ml collagenase type I (Invitrogen, Carlsbad, of WT:CD28ko cells was determined by flow cytometry. Mice were then CA), and 200 U/ml hyaluronidase (SERVA Electrophoresis, Heidelberg, infected with MCMV or LCMV Cl13 as indicated. Germany) for 30 min at 37˚C and then mechanically dissected to generate Real-time RT-PCR. Total RNA was extracted using RNeasy kits (Qiagen, a single-cell suspension. Cells were stained with 10 mg/ml anti–PDCA-1 Redwood City, CA), digested with DNase I (RNase-free DNase set; Qia- allophycocyanin (JF05-1C2.4.1; Miltenyi Biotec, Auburn, CA), anti– gen), and reverse-transcribed into cDNA using Superscript III RT (Invi- CD11c-PE (HL3), anti–B220-FITC (RA3-6B2; BD Biosciences, San trogen). cDNA quantification was performed using SYBR Green PCR kits Diego, CA), and anti–CD45-PerCP (30-F11; eBioscience, San Diego, and a Real-Time PCR Detection System (Applied Biosystems, Carlsbad, CA). Cells were acquired on a FACSCalibur (BD Biosciences, San Jose, CA). The relative transcript levels were normalized against Gapdh as CA) and analyzed using FlowJo software (Tree Star, Ashland, OR). described previously (22). The following primers were used: Ifna primers

recognizing Ifna 4 and 6,59-TATGTCCTCACAGCCAGCAG-39 (forward) by guest on September 29, 2021 Human samples and cell lines and 59-TTCTGCAATGACCTCCATCA-39 (reverse); Ifnb1,59-CTGGCT- TCCATCATGAACAA-39 (forward) and 59-GAGGGCTGTGGTGGA- Residual axillary lymph node samples (n = 5), following diagnostic studies, GAA-39 (reverse); Cd28,59-ACAGTTGGGCCACTTGTTGTCCTTT-39 were used for these studies where samples were incubated with RPMI 9 9 Mx1 1640 medium + collagenase (1 mg/ml; Roche, Indianapolis, IN) for 20 min (forward) and 5 -GCTCCCAATGGTGCCTTCTGGA-3 (reverse); , 9 9 9 at 37˚C and passed through a 100-mm strainer to achieve a single-cell 5 -CAACTGGAATCCTCCTGGAA-3 (forward) and 5 -GGCTCTCCTC- 9 Rig-I Ddx58 9 suspension. Cal-1 cells were provided by Dr. S. Kamihira (Nagasaki AGAGGTATCA-3 (reverse); recognizing ,5-CGGGACC- 9 9 University Graduate School of Biomedical Sciences, Nagasaki, Japan). CACTGCCTCAGGT-3 (forward) and 5 -GCATCCAGGGCGGCACA- GAG-39 (reverse); Tnfa,59-CCCTCACACTCAGATCATCTTCT-39 (forward) PBLs (n = 7 donors) were isolated using standard Ficoll gradient separa- and 59-GCTACGACGTGGGCTACAG-39 (reverse); and MCMV eI,59- tion as described previously (35). Cells were stained with CD16 (3G8), 9 9 CD56 (MEM-188), CD14 (HCD14), CD19 (HIB19)-Pacific Blue, CD3 GAGTCTGGAACCGAAACCGT-3 (forward) and 5 -GTCGCTGTTAT- 9 Il6 allophycocyanin Cy7 (UCHT1), B220 Percp Cy5.5 (RA3-6B2), HLADR CATTCCCCAC-3 (reverse). Transcript levels of were determined rel- Gapdh PE Cy7 (L243), CD11c Alexa 488 (3.9), and CD123 allophycocyanin ative to using primer and probe sets from the Universal Probe Library (6H6) (BioLegend, San Diego, CA). pDCs were identified as Lineage (Roche). Cytokine transcript levels were measured 6 h poststimulation. 2 + 2 + (CD16, CD56, CD14, CD19, B220, CD3) HLADR CD11c CD123 . Microarray T cells were identified as CD3+. T cells and pDCs (lymph node biopsies) or Cal-1 pDCs were stained for CD28 expression with CD28 PE (CD28.2) RNA extracted from FACS-purified splenic pDCs, CD8+ DCs, and CD11b+ or isotype control (mouse IgG1) (BioLegend) and acquired on a BD LSRII DCs from uninfected WT mice were used for DNA microarray using and analyzed using FlowJo software. GeneChip mouse genome 430 2.0 arrays (Affymetrix, Santa Clara, CA). Differential gene expression was determined as fold of change of indicated Purification of BM and spleen DCs genes over background intensity. Microarray data have been deposited in BM cells were isolated from femurs and tibias, and a single-cell suspension the National Center for Biotechnology Information’s Gene Expression Omnibus (38) under the accession number GSE75834 (https://www.ncbi. was prepared and cultured 7–8 d in the presence of 100 ng/ml Flt3 ligand nlm.nih.gov/geo/query/acc.cgi?acc=GSE75834). (Flt3L) (Amgen, Thousand Oaks, CA; Cell Dex Therapeutics, Needham, MA) as described previously (36). Spleens were incubated with 1 mg/ml Generation of CD28 retrovirus construct (RVGFP-CD28) collagenase D for 20 min at 37˚C and passed through a 100-mm strainer to achieve a single-cell suspension. Splenocytes were enriched with PanDC CD28 cDNA clone (MGC premier cDNA; TransOMIC Technologies, microbeads using an Automacs system (Miltenyi Biotec). PanDC+ frac- Huntsville, AL) was amplified by PCR, where XhoI and HpaI restriction tions were stained with propidium iodide (PI) and FACS-purified using a sites were incorporated using primers: 59-GACTCGAGGCCGCCAC- BD ARIA II (BD Biosciences) for pDCs (PI2CD11cintermediate/dimCD11b2 CATGACACTCAGGCTGCTGTTCTTGG-39 (forward) and 59-TCGTTAACT- B220+PDCA+) and CD11b+ cDCs (PI2CD11c+B2202CD11b+) after B CAGGGGCGGTACGCTGCAAAGT-39 (reverse). The amplicon was gel- (CD19), T (Thy1.2), and NK (Nk1.1) cell exclusion. BM-pDCs and BM- purified and digested with XhoI and HpaI restriction enzymes, according cDCs were stained and sorted as PI2CD11c+CD11b2B220+PDCA+ and to the manufacturer’s instructions (New England Biolabs, Ipswich, MA), and PI2CD11c+B2202CD11b+, respectively. Purity of the cells was .92%. ligated to previously digested pMIGR (39) (GFP-labeled retroviral vector; Cells were stimulated with CpG B 1668 (Integrated DNA Technologies, San RVGFP) (Addgene, Cambridge, MA). 293T cells were transfected with Diego, CA) at 0.1 mM (BM-derived DC) or 1 mM (splenic DC), 10 mMCpG RVGFP-CD28 or empty vector for control, LT1 transfection reagent (Mirus A 2336 (InvivoGen, San Diego, CA), and 100 mM loxoribine (InvivoGen). Bio, Madison, WI), and the pcl-Eco packaging plasmid required to produce 1902 CD28 INHIBITS pDC IFN PRODUCTION viral vectors. Seventy-two hours later, supernatants were harvested and pDCs (data not shown), it was consistently expressed above isotype stored at 280˚C until transfection of pDCs. levels in pDCs obtained from lymph node biopsies (Fig. 1F, CD28 overexpression by retrovirus. At day 3 post-Flt3L culture, CD28ko Supplemental Fig. 1C). Altogether, these data indicated that the BM cells were transduced with RVGFP-CD28 or empty vector control prototypic T cell costimulatory molecule CD28 is constitutively (RVGFP) and polybrene reagent (Fisher Scientific) and spin-infected room temperature at 1000 3 g at 90 min. Cells were incubated 37˚C overnight. expressed in pDCs. On day 4 post-Flt3L culture, cells were again transduced with RVGFP- CD28 or RVGFP and incubated 3 h at 37˚C. Cells were then washed in CD28 downregulates pDC cytokine production upon TLR PBS and placed in fresh DC medium + Flt3L. On day 8 postculture, cells stimulation were harvested and FACS-purified for GFP+ pDC fractions. Purified cells were stimulated with 0.1 mM CpG B 1668 6 h and harvested for mRNA de- To investigate a putative role for CD28 in pDC differentiation, tection of IFN-I and proinflammatory cytokine transcripts relative to Gapdh. survival, and/or function, we first examined pDCs obtained from WT versus CD28ko BM-Flt3L cultures. Similar percentages and Statistics numbers of live pDCs were obtained at day 7–8 postculture, in- Unpaired Student t tests or ANOVA tests were performed using GraphPad dicating normal pDC and cDC development from BM progenitors Prism software (GraphPad, La Jolla, CA). Error bars represent mean 6 (Supplemental Fig. 2A, 2B). In contrast, significantly higher levels , SEM. A p value 0.05 was considered statistically significant. of IFN-I were detected upon stimulation of FACS-purified Study approval CD28ko BM-derived pDCs with the TLR7 agonist loxoribine or TLR9 agonist CpG (Supplemental Fig. 2C). We next sought to Human axillary lymph node biopsies sent to the University of California San Diego Clinical Flow Cytometry Laboratory were deidentified and used for extend these findings to pDCs freshly isolated from spleens of WT research, according to University of California San Diego Institutional and CD28ko mice. Consistent with normal development of DCs in Downloaded from Review Board–approved protocol number 130973x. Peripheral blood the absence of CD28, we observed comparable percentages and samples were collected from healthy volunteers (written informed consent numbers of pDCs and cDCs in WT and CD28ko spleens (Fig. 2A was obtained prior to study inclusion) at the University of California San Diego CFAR Clinical Investigation Core Antiviral Research Center, and data not shown, respectively). Importantly, as described for according to University of California San Diego Institutional Review BM-Flt3L cultures, significantly increased levels of IFN-I were Board–approved protocol number 110522. Mice were bred and maintained detected when FACS-purified splenic pDCs from CD28ko mice in a closed breeding facility and mouse handling conformed to the re- were stimulated with loxoribine, CpG A, or CpG B. Moreover, http://www.jimmunol.org/ quirements of the National Institutes of Health and the Institutional Animal quantification of TNF-a and IL-6 in these same culture conditions Care and Use Guidelines of University of California San Diego, according to approved protocol S07315. revealed a similarly increased production of these proinflammatory cytokines by CD28ko compared with WT pDCs, whereas viability Results of both WT and CD28ko pDCs was similar following agonist stimulation (Fig. 2B). Despite changes in cytokine response, CD86 CD28 is constitutively expressed in pDCs (a hallmark of DC maturation (46)) and MHC-II were equally Previous analyses have demonstrated that, although pDCs and cDC expressed in WT versus CD28ko pDCs before and after TLR subsets (including CD8+ and CD11b+ cDCs) are derived from a stimulation, although a minor increase in MHC-II was observed in distinct branch of the leukocyte family tree and exhibit functional loxoribine-treated CD28ko pDCs (Fig. 2C). Furthermore, although by guest on September 29, 2021 differences, they maintain an evolutionarily conserved transcrip- incubation of murine WT pDCs and human Cal-1 pDCs with tional signature (40, 41). Therefore, we surmised that a compar- agonistic anti-CD28 Ab or rCTLA4-Ig (47, 48) showed no effect ison between pDCs and cDCs may highlight regulatory molecules on cytokine production (data not shown), we did observe reduced that selectively modulate pDC function. We noted that the ex- Ifna in CD28ko pDCs upon reconstitution of CD28 levels (Fig. 2D), pression of the gene encoding the prototypic T cell costimulatory ruling out any off-target effects in IFN-I production by CD28ko molecule CD28 was 59 and 57 times higher in pDCs compared pDC. The levels of Tnfa and Il6 transcripts were, however, un- with CD11b+ and CD8+ cDCs, respectively (Fig. 1A). Other CD28 changed in CD28ko pDCs with restored CD28 expression, raising family receptors (42) were either undetectable in pDCs (i.e., Ctla4, the possibility that the effect of CD28 may be more profound and/or Pdcd1,andIcos) or equally expressed in all DC subsets (i.e., Btla). selective for IFN-I than for proinflammatory cytokines. Notably, DC subset or T cell–specific gene transcripts were selec- Given that CD80 and CD86 engage CD28 to promote T cell tively expressed or absent, respectively. To confirm CD28 expres- activation and are upregulated in pDCs upon TLR stimulation sion in pDCs, we first determined Cd28 transcript levels by (Fig. 2C) (8–10), we next evaluated their putative role in pDC quantitative PCR (qPCR) in both murine BM–derived DCs 7 d IFN-I production. Interestingly, FACS-purified pDCs from CD80/ postculture with Flt3L and splenic DC subsets. We observed that 86 dko BM-Flt3L cultures exhibited enhanced IFN-I production in Cd28 transcripts were undetectable in cDCs but were significantly response to loxoribine and CpG stimulation when compared with expressed in both BM-derived and splenic pDCs, albeit to a lesser WT BMpDCs (Fig. 2F), although this effect was not observed extent than in splenic T cells (Fig. 1B, Supplemental Fig. 1A, 1B). with FACS-purified splenic pDCs from CD80/86 dko mice (data Surface expression of CD28 was also present in pDCs not shown), possibly because of other anomalies in CD80/86 dko freshly obtained from spleen, BM, blood (Fig. 1C), and BM-Flt3L mice. Altogether, these data demonstrated that, in the absence of cultures (Fig. 1D), with levels lower than in T cells but contrasting CD28, pDC development and maturation were unchanged, but the background expression in cDCs. Of note, basal surface and IFN-I production was significantly increased in response to TLR intracellular CD28 expression were similar, and CD28 expression stimulation, revealing a novel regulatory role for CD28 in limiting did not increase in response to TLR stimulation (data not shown). the magnitude of pDC cytokine responses. Furthermore, our data Finally, we examined CD28 protein expression in human pDCs. suggest that CD80/CD86 molecules, which are natural CD28 li- For this, we first analyzed the Cal-1 human pDC cell line (43) and gands, may also mediate IFN-I downregulation in pDCs. compared it with non-pDC human cell lines A549 (lung epithelial cells) (44) and Thp-1 (monocytic cells) (45). Similar to murine CD28 limits pDC-derived IFN-I and antiviral defense early pDCs, CD28 expression was observed in Cal-1 but not A549 or after in vivo viral infection Thp-1 cells (Fig. 1E). Furthermore, analysis of primary human We next investigated the effect of CD28 signaling on pDC IFN-I pDCs revealed that, although CD28 was undetectable in blood response and host defense to viral infection in vivo. We first in- The Journal of Immunology 1903 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. pDCs constitutively express CD28. (A) Splenic pDCs, CD11b+cDCs, and CD8a+cDCs were FACS-purified from WT mice and processed for DNA microarray analysis. Heat map depicts fold of change (FC) of indicated genes over background. (B) Cd28 expression relative to Gapdh was de- termined in pDCs and cDCs FACS-purified from BM cultured for 8 d in the presence of Flt3L and from spleens. CD4+ T cells (Thy1.2+CD4+) and CD8+ T cells (Thy1.2+CD8+) purified from spleens were also evaluated. Statistical analysis performed between pDCs and other cell types. (C and D) Surface expression of CD28 was measured by flow cytometry in WT murine T cells, pDCs, and cDCs from spleen, BM, and blood ex vivo (C) and BM 8 d post- Flt3L cultures (D). (E and F) CD28 expression was evaluated in human cell lines A549 (lung epithelial cells), Cal-1 (pDCs), and Thp-1 (monocytes) (E) and T cells (CD3+) and pDCs (Lineage2HLADR+CD11c2CD123+) from human axillary lymph nodes (F). Representative histograms of CD28 expression (black open) overlaid with isotype (gray filled) are shown. Bar graphs depict mean values 6 SEM of mean fluorescence intensity (MFI) for CD28. Data are derived from one experiment with cells pooled from 5 to 10 mice (A) or are representative of four to six independent experiments with n = 3–5 mice/group (B–D) or from n = 3 independent experiments of cells lines (E) and n = 5 human lymph node samples (F). *p , 0.05, **p , 0.01, ***p , 0.001. fected WT and CD28ko mice with LCMV Cl13, an ssRNA virus in To further validate the inhibitory role of CD28 on pDC antiviral which pDCs, via TLR7, contribute to peak IFN-I response at 24 h function in vivo and to examine its biological relevance in a model postinfection (p.i.) (20, 22). At this time point postinfection, we where the contribution of pDCs to viral control is well established, detected higher IFN-I in serum from CD28ko versus WT mice we next evaluated WTand CD28ko mice infected with MCMV (11, infected with LCMV, whereas no difference in systemic TNF-a 12). During MCMV infection, pDCs recognize and respond to levels was observed (Fig. 3A, 3B). Importantly, splenic pDCs viral infection in a TLR9-dependent manner and are essential for isolated at this same time point from LCMV-infected CD28ko the systemic IFN-I peak observed at 36 h p.i. (11, 12). We ob- mice showed significantly higher levels of Ifna and Ifnb tran- served that, although systemic IFN-I was undetectable in WT and scripts compared with pDCs from WT infected mice, whereas CD28ko mice at 24 h p.i., they were enhanced at 36 h p.i. and that cDCs from both WT and CD28ko mice demonstrated low to un- this elevation was significantly higher in CD28ko compared with detectable IFN-I levels, as described previously (Fig. 3C) (20). WT infected mice (Fig. 3D), whereas serum TNF-a levels were However, we observed no differences in LCMV Cl13 replication similar between both groups (Fig. 3E). Of note, at 36 h p.i., pDC in CD28ko versus WT pDCs or liver homogenates at day 1 p.i. numbers were similar between WT and CD28ko mice, demon- (data not shown), potentially because of abundant and sufficient strating that the difference in systemic IFN-I levels was not the IFN-I already present at this time point in WT controls. result of increased numbers of pDCs in CD28ko mice (data not 1904 CD28 INHIBITS pDC IFN PRODUCTION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. CD28 downregulates pDC IFN-I production upon TLR stimulation. (A) Graphs depict the proportion and number of splenic pDCs from WT and CD28ko mice where mice are plotted individually and mean 6 SEM are shown. (B and C) Splenic pDCs were FACS-purified and stimulated with loxoribine, CpG A, CpG B, or medium alone for 15 h. Cells were analyzed for viability poststimulation, and supernatants were analyzed for IFN-I bioactivity by bioassay and TNF-a and IL-6 levels by ELISA (B). Cells were analyzed for CD86 and MHC-II expression by flow cytometry. Representative histograms are shown where WT (black open) and CD28ko (gray open) pDCs are overlaid. Bar graphs depict mean fluorescence intensity (MFI) for CD86 and MHC-II (C). (D and E) Total BM cells from CD28ko mice were transduced with retroviral constructs containing CD28 overexpression plasmid (RVGFP-CD28) or empty vector control (RVGFP). Retroviral-transduced (GFP+) or untransduced (GFP2) pDCs were analyzed at day 8 post-Flt3L culture and analyzed for CD28 protein expression by flow cytometry. FACS plot depicts proportion of pDCs transduced by RVGFP-CD28, indicated by GFP+ cells. Representative histograms are shown where GFP+ pDCs (retrovirus incorporated; green open) are overlaid with GFP2 pDCs (retrovirus not incorporated; black open) for RVGFP and RVGFP-CD28 and shown with respect to CD28 isotype control (gray filled) of RVGFP-CD28. Bar graph depicts CD28 mean fluorescence intensity (D). FACS-purified GFP+ pDCs were stimulated with CpG B for 6 h and evaluated for Ifna, Tnfa, and Il6 relative to Gapdh by qPCR (E). (F) BM from WT and CD80/86 dko mice was cultured in Flt3L for 7–8 d, and pDCs were FACS-purified and stimulated with loxoribine, CpG B, or medium alone 15 h. Supernatants were analyzed for IFN-I bioactivity by bioassay. Data are representative of two to four independent experiments with three to six mice per group. Bar graphs depict mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. shown). Consistently, FACS-purified splenic pDCs isolated from the absence of CD28, we detected lower viral replication, as CD28ko MCMV-infected mice 36 h p.i. demonstrated higher indicated by decreased numbers of PFU and transcript levels levels of Ifna and Ifnb transcripts than their WT counterparts of the MCMV early inducible (eI) gene in the livers of (Fig. 3F). As expected, undetectable levels of Ifna and Ifnb CD28ko compared with WT infected mice (Fig. 3H). Taken transcripts were observed in WT and CD28ko cDCs at 36 h p.i. together, these results indicate that CD28 inhibited pDC and (Fig. 3F) (36). Consistent with the enhanced IFN-I response in systemic IFN-I production during in vivo viral infection with CD28ko mice, expression of prototypic ISGs (i.e., Mx1 and RigI) both RNA (LCMV) and DNA (MCMV) viruses. Furthermore, were upregulated in both pDCs and cDCs from CD28ko com- this effect was associated with a restricted antiviral state and pared with WT MCMV-infected mice (Fig. 3G). Importantly, in influenced viral control early after MCMV exposure, support- The Journal of Immunology 1905 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021 FIGURE 3. CD28 restricts IFN-I production and early virus control during in vivo viral infection. (A–C) WT and CD28ko mice were infected with LCMV Cl13 for 24 h. Serum IFN-I and TNF-a levels were evaluated by bioassay (A)andELISA(B), respectively, and FACS-purified splenic pDCs and cDCs were evaluated for Ifna and Ifnb transcript levels relative to Gapdh by qPCR (C). (D–H) WT and CD28ko mice were infected with MCMV for36h.SerumIFN-IandTNF-a levels were determined at 24 and 36 h p.i. by bioassay (D) and ELISA (E), respectively. FACS-purified splenic pDCs and cDCs were evaluated by qPCR for Ifna, Ifnb (F), Mx1,andRigI (G) transcript levels relative to Gapdh. MCMV titers were quantified by plaque assay and depicted as PFU per gram (g) of tissue, and expression of MCMV eI gene relative to Gapdh wasdeterminedbyqPCRinlivers(H). Bar graphs show mean value 6 SEM. Data are representative of two to three independent experiments with four mice per group. *p , 0.05, **p , 0.01, ***p , 0.001. ing the biologically relevant role of CD28 on pDC IFN-I re- expression of the MCMV eI gene was greatly downregulated in sponse. pDCs (but not cDCs) from the CD28ko origin compared with their WT counterparts, indicating that intrinsic CD28 signaling (po- Cell-intrinsic CD28 signaling suppresses pDC-IFN-I response tentially through suppression of an autocrine IFN-I effect) was upon in vivo viral exposure promoting MCMV replication in pDCs (Fig. 4F). Altogether these To investigate whether the inhibitory effect of CD28 on pDC-IFN-I data indicate that cell-intrinsic CD28 signaling on pDCs signifi- production during viral infection was cell-intrinsic or resulted from cantly downregulated their IFN-I and proinflammatory cytokine CD28 deficiency on other cell types, we generated WT:CD28ko production in response to in vivo RNA and DNA viral infections. mixed chimeras (Fig. 4A). Chimeric mice showed similar pro- In the case of MCMV infection, cell-intrinsic CD28 signaling also portions of total lymphocytes, pDCs, and cDCs in WT versus resulted in a restricted antiviral state and increased viral replica- CD28ko compartments (Supplemental Fig. 3). Notably, pDCs tion, indicating that CD28 signaling directly regulated pDC anti- isolated from LCMV-infected WT:CD28ko mixed chimeric mice viral defense during in vivo infection. 24 h p.i. showed enhanced expression of Ifna and Ifnb transcripts when derived from the CD28ko compared with WT origin (Fig. CD28 deficiency enhances the IFN-I signature in a wound 4B). Moreover, 36 h after MCMV infection, FACS-purified healing model splenic pDCs from the CD28ko compartment showed an even Aside from its role in antiviral defense, pDC IFN-I production also more dramatic increase of Ifna and Ifnb expression compared promotes innate defense during tissue injury (31, 49). Indeed, pDCs with WT pDCs (Fig. 4C). Similarly, proinflammatory cytokine sense host-derived nucleic acids that are released following common levels were elevated in CD28ko pDCs compared with their WT skin injuries, migrate to the site of cutaneous lesion, and secrete counterpart (Fig. 4D). Interestingly, pDC Mx1 expression was also IFN-I, promoting tissue re-epithelialization and wound repair (31). enhanced in CD28ko pDCs during MCMV infection, suggesting To investigate the role of CD28 in pDC IFN-I response triggered autocrine IFN-I signaling in this setting (Fig. 4E). Furthermore, by tissue injury, we compared pDC infiltration and IFN-I levels in 1906 CD28 INHIBITS pDC IFN PRODUCTION Downloaded from http://www.jimmunol.org/

FIGURE 4. Cell-intrinsic CD28 signaling limits pDC cytokine production and their antiviral defense during in vivo viral infection. (A–F) CD45.1+ WT mice were sublethally irradiated, reconstituted with a 50:50 ratio of CD45.1+ WT and CD28ko (CD45.2+) BM cells for 8 wk (A) and infected with LCMV Cl13 for 24 h (B) or MCMV for 36 h (C–F). (B–E) Expression of Ifna, Ifnb (B and C), Tnfa, Il-6 (D), Mx1 (E), and MCMVeI gene (F) relative to Gapdh were determined by qPCR in splenic FACS-purified pDCs and cDCs. Bar graphs show mean value 6 SEM. Data are representative of two independent ex- by guest on September 29, 2021 periments with four to five mice per group. *p , 0.05, **p , 0.01, ***p , 0.001.

WT and CD28ko mice following mechanical injury where the upper in vivo, indicating that it is a general mechanism that down- epidermal layer of the skin is removed (50). No differences were regulates pDC innate response in different immune settings. observed in pDC infiltration between WT and CD28ko mice at 24 or CD28 costimulation in T cells, through interaction with CD80 48 h postinjury, the time at which pDC infiltrate peaks (data not and/or CD86 molecules on APCs, is one of the best established shown and Fig. 5A, respectively). Interestingly, despite no change in events that bridge innate and adaptive immunity following path- pDC number, CD28ko mice demonstrated an enhanced IFN-I sig- ogen recognition (32, 51). Interestingly, CD28 expression has been nature, which in this model, is known to be fully dependent on pDCs previously reported in other immune cells including NK cells, (31). In fact, 48 h postinjury, we observed increased transcript levels eosinophils, neutrophils, and plasma cells. For example, engage- of Ifna5 and Ifna6 (Fig. 5B) and the IFN-I response gene Ifi202b. ment of CD28 on a subset of NK cells promotes activation and Irf7 and Isg15 also trended toward increased levels in CD28ko mice, NK-mediated cytotoxicity (52), whereas CD28 activity on eosin- but differences did not reach statistical significance (Fig. 5C). These ophils promotes their activation and secretion of IL-2, IFN-g, and data indicate that CD28 suppression of pDC IFN-I response was not IL-13 (53, 54). Furthermore, CD28 ligation on neutrophils en- restricted to viral infection but instead also influenced nonviral innate hances CXCR-1 expression, promoting their migration (55), and responses such as those following tissue injuries. also induces neutrophil IFN-g secretion during Leishmania major infection (56). More recently, plasma cells were found to express Discussion CD28 (57), but the role of CD28 in regulating plasma cell survival pDCs are highly specialized to produce IFN-I, which dramatically and function is unclear as evidence for both promoting and lim- influence antiviral and anticancer defense, autoimmunity, and iting plasma cell survival and Ab production have been reported wound healing, among other human illnesses (1). Therefore, un- (58, 59). Importantly, our in vivo experiments with BM-mixed derstanding the regulation of pDC IFN-I production at the mo- chimeras indicated that CD28 dampened pDC function and its lecular level is of great importance to ultimately fine-tune the antiviral state, promoting viral replication in a cell-intrinsic magnitude of IFN-I responses as well as their multiple (and often manner, rather than through signaling via the aforementioned opposing) biological consequences (1). We found that pDCs non-pDC cells that also express CD28. This is consistent with the constitutively expressed the prototypic T cell stimulatory mole- enhanced cytokine production in CD28ko pDCs differentiated in cule CD28, which unexpectedly restrained (rather than stimulated) BM-Flt3L cultures where pDCs are the only CD28-expressing pDC IFN-I production in response to TLR stimulation. Impor- cells. Of note, reconstitution of CD28 in CD28ko pDCs from tantly, CD28-mediated pDC suppression was also observed upon such BM-Flt3L cultures downregulated their IFN-I production, infection with RNA and DNA viruses and following skin injury ruling out any off-target effects in IFN-I production by CD28ko The Journal of Immunology 1907

FIGURE 5. CD28 deficiency enhances pDC IFN-I signature in response to skin injury. Skin injury was induced in WT and CD28ko mice by tape stripping (tape stripped) or left uninjured (No Tx) and evaluated 48 h postinjury. (A) Dermal cell suspensions were isolated from uninjured and injured skin and viable pDCs were counted (CD11c+B220+PDCA+). Repre- sentative FACS plots are shown for injured mice. (B and C) Transcript levels of Ifna2, Downloaded from Ifna5, and Ifna6 (B), and Ifi202b, Irf7, and Isg15 (C) were determined relative to Gapdh by qPCR. Graphs depict mean 6 SEM, where symbols represent individual mice. Data are representative of two inde- pendent experiments with four to five mice , , , per group. *p 0.05, **p 0.01, ***p http://www.jimmunol.org/ 0.001. by guest on September 29, 2021 pDCs. Overall, it is striking that although CD28 appears to induce molecule lymphocyte activating gene 3 (61, 62) also controls the stimulatory signals that promote activation, enhanced function, homeostatic proliferation and expansion of pDCs (63). Taken to- and/or survival in most cells that express it, it exerts a suppressive gether with CD28, these and other homeostatic pDC regulators role in pDCs. It is tempting to speculate that such opposing roles such as E2-2 (64), Flt3 (65), and the PI3k/mTOR pathway (66) of CD28 in pDCs versus other immune cells might have evolved highlight the important and intricate nature of pDC control under to counterbalance the immune response to best fight infections steady state, where these multiple pathways may partially overlap with minimal collateral tissue damage. but may also regulate distinct aspects of pDC biology, pre- Interestingly, our findings suggest the possibility that CD80/ conditioning subsequent pDC responses. CD86 interaction with CD28 could regulate the ability of pDCs Although CD28 signaling in pDCs may be critical for homeo- to produce IFN-I. Because we observed enhanced pDC-IFN-I static pDC function, it also holds the potential for therapeutic production in FACS-purified CD80/86 double-deficient BM- exploitation to limit excessive IFN-I responses that contribute to pDCs, it is possible that CD80/CD86 can act in a pDC- autoimmunity or immunopathology. In fact, in several autoimmune autonomous manner. However, it is also conceivable that other diseases, pDCs play a pathogenic role via TLR recognition of self CD80/86-expressing cells (such as cDCs) might also modulate nucleic acids, resulting in excessive IFN-I production that promotes pDC IFN-I production via CD28 signaling. Moreover, although it is activation and survival of autoreactive T and B cells (26, 27, 29, 30, still unclear at what stage of the pDC lifespan CD28 suppression 67). Given that CD28 promotes activation of conventional T cells becomes effective, failure to alter IFN-I production by ex vivo while also expanding regulatory T cells and dampening pDC in- CD28 stimulation or blockade in fully differentiated WT pDCs nate responses, it is not surprising that the effect of ubiquitous (concomitantly or 1–2 h before TLR stimulation) suggests that CD28 inhibition or stimulation varies in different autoimmune CD28 may homeostatically regulate pDCs (i.e., before pDCs en- disease settings. Indeed, although studies have shown that CD28- counter pathogen-associated molecular patterns). Similarly, a re- mediated signaling prevents spontaneous development of auto- cent study demonstrated homeostatic regulation of pDCs by the immune diabetes (68) and reduces experimental autoimmune microRNA miR-126, which (in steady-state conditions) is re- neuritis (69), others have shown that CD28 promotes autoimmune quired for pDC survival and optimal expression of molecules in- diseases such as collagen-induced arthritis, experimental autoim- volved in pDC TLR responses, affects subsequent pDC antiviral mune encephalomyelitis, and systemic lupus erythematosus (70– responses (60). Interestingly, miR-126 regulates pDCs through the 72). Although these studies have primarily focused on the effects VEGFR2 protein, which, similar to CD28, is undetectable in hu- of CD28 on conventional or regulatory T cells, our results open man pDCs isolated from blood but significantly expressed in pDCs the possibility that altered IFN-I production by pDCs may also purified from tissues (60). Furthermore, the T cell regulatory play a role in the abovementioned autoimmune disease models. 1908 CD28 INHIBITS pDC IFN PRODUCTION

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