Chronic Viral Infection in Mice Ligand Expression on APC Subsets Over

Conserved and Differential Features of TNF Superfamily Ligand Expression on APC Subsets over the Course of a Chronic Viral Infection in Mice Kuan C. Wang, Kuan-Lun Chu, Nathalia V. Batista and Tania H. Watts Downloaded from ImmunoHorizons 2018, 2 (11) 407-417 doi: https://doi.org/10.4049/immunohorizons.1800047 http://www.immunohorizons.org/content/2/11/407 http://www.immunohorizons.org/ This information is current as of October 6, 2021. Supplementary http://www.immunohorizons.org/content/suppl/2018/12/20/2.11.407.DCSup Material plemental References This article cites 33 articles, 10 of which you can access for free at: http://www.immunohorizons.org/content/2/11/407.full#ref-list-1 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: by guest on October 6, 2021 http://www.immunohorizons.org/alerts ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732. RESEARCH ARTICLE Infectious Disease Conserved and Differential Features of TNF Superfamily Ligand Expression on APC Subsets over the Course of a Chronic Viral Infection in Mice Kuan C. Wang, Kuan-Lun Chu, Nathalia V. Batista, and Tania H. Watts Downloaded from Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada ABSTRACT http://www.immunohorizons.org/ There is currently much interest in how different APC subsets shape the immune response. We recently described a division of labor between classical dendritic cells (cDC) and inflammatory monocyte-derived APC in provision of costimulatory ligands to T cells early during chronic lymphocytic choriomeningitis clone 13 (LCMV 13) infection in mice. At day 2 of LCMV 13 infection, cDC preferentially express CD80 and CD86, whereas TNF superfamily ligands GITRL, 4-1BBL, CD70, and OX40L are preferentially induced by type I IFN on inflammatory monocyte-derived APC, with minimal expression on cDC. In this study, we further investigate the expression of TNF and B7 family ligands on APC over the course of LCMV 13 infection. OX40L and 4-1BBL remain above baseline through the chronic stage of infection, with predominant expression on inflammatory APC compared with cDC in the spleen, partially blocked by anti–IFN-gR Ab pretreatment. Conversely, CD70, like GITRL, returns to baseline on the APC within a few days postinfection. In the lung, TNF family ligands were also preferentially expressed on inflammatory monocyte-derived APC. CD86 was generally higher on cDC than inflammatory APC in the spleen, but in the lung CD86 was highest on inflammatory APC. Moreover, in the spleen, CD80 by guest on October 6, 2021 levels on different APC subsets fluctuated over the course of the infection. We also show that LPS induction of TNF superfamily ligands is largely mediated through type I IFN. This study highlights the importance of IFNs and monocyte-derived APC in TNF superfamily ligand expression in both secondary lymphoid organs and tissues during chronic viral infection. ImmunoHorizons, 2018, 2: 407–417. INTRODUCTION of the TNF superfamily contribute to T cell activation by providing survival signals to sustain T cell accumulation (4, 5). Recently, our T cell priming requires the recognition of peptide–MHC by the laboratory provided evidence that there is a division of labor Ag-specific TCR (signal 1) as well as corecognition of B7 family between different APC subsets in providing costimulatory costimulatory ligands (CD80, CD86) binding to CD28 (signal 2) (1). molecules to T cells (6). We showed that at day 2 of lymphocytic Cytokines produced early during infection also play a pivotal role choriomeningitis (LCMV) clone 13 infection in the spleen or day 3 in induction of the costimulatory ligands as well as in providing of influenza A infection in the draining lymph node, cell-specific signals for T cell differentiation and expansion (signal 3) (2, 3). In effects of type I IFN (IFN-I) resulted in a dichotomy of expression addition to the B7 family of costimulatory ligands, several members of TNF versus B7 superfamily ligands on different APC subsets. Received for publication July 11, 2018. Accepted for publication November 29, 2018. Address correspondence and reprint request to: Dr. Tania H. Watts, University of Toronto, Department of Immunology, Medical Sciences Building, Room 7221, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada M5S 1A8. E-mail address: [email protected] ORCID: 0000-0001-7897-4890 (T.H.W.). This work was supported by Canadian Institutes of Health Research (CIHR) Foundation Grant FDN 143250 (to T.H.W.). K.C.W. was funded by a CIHR Canada Graduate Scholarship Masters award and K.-L.C. by an Ontario Graduate Scholarship. T.H.W. holds the Sanofi Pasteur Chair in Human Immunology at the University of Toronto. Abbreviations used in this article: cDC, classical dendritic cell; DC, dendritic cell; IFN-I, type I IFN; InfAPC, inflammatory monocyte-derived APC; LCMV, lymphocytic choriomeningitis; MHC II, MHC class II; pDC, plasmacytoid DC; p.i., postinfection; TG, thioglycolate-elicited. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license. Copyright © 2018 The Authors https://doi.org/10.4049/immunohorizons.1800047 407 ImmunoHorizons is published by The American Association of Immunologists, Inc. 408 COSTIMULATORY LIGAND EXPRESSION DURING CHRONIC INFECTION ImmunoHorizons Classical dendritic cells (cDC), including dendritic cell (DC) 1 and described (12). A total of 2 3 106 focusformingunitsofLCMV13 DC2, were found to have the highest expression of MHC class II were injected i.v. per mouse where indicated. (MHC II), CD80, and CD86 at day 2 after viral infection, whereas CD64+FceRI+ inflammatory monocyte-derived APC (InfAPC) had Mice low levels of MHC II, CD80, and CD86 but high levels of the TNF Age-matched (6–10 wk old) CD45.2 C57BL/6NCrl female mice family ligands 4-1BBL, GITRL, CD70, and OX40L. Using the GITR were used in all experiments. All animals were housed under costimulatory pathway for proof of concept, we went on to show specific pathogen-free conditions in the Division of Comparative that GITRL on InfAPC provided a postpriming checkpoint, which Medicine at the Terrence Donnelly Centre for Cellular and we called signal 4, that allowed T cells to survive and accumulate to Biomolecular Research (University of Toronto). Animal experi- help control chronic LCMV infection (6). ments were approved by the animal care committee at the Our previous studies of costimulatory ligand expression on University of Toronto in accordance with the Canadian Council on APC subsets during viral infection focused on the first 2 to 3 days of Animal Care (Protocol no. 20011642). infection and on responses to LCMV 13 in the spleen (6). However, T cells can be recruited into an immune response throughout the Abs and reagents course of infection (7). Moreover, APCs are dynamically regulated All Abs and related reagents used in this study are defined in Downloaded from over the course of infection (8, 9), with rapid turnoverof some APC Table I. populations (10). For this reason, it is important to understand APC subset-specific costimulatory molecule expression through to the Flow cytometry chronic phase of infection, as well as to analyze costimulatory Freshly isolated spleens were processed through 70-mm cell molecule expression in the tissues as well as the secondary strainers to create single-cell suspensions. Following perfusion, http://www.immunohorizons.org/ lymphoid organs. Throughout the course of LCMV 13 infection, as lung tissues were first digested with collagenase IV and DNase I for expected, MHC II was found to be consistently higher on cDC 45 min at 37°C on a shaker prior to the use of cell strainers. Lung compared with InfAPC, whereas InfAPC consistently express the tissue was then mechanically disrupted through a 70-mm cell highest levels of TNF family ligands compared with cDC or strainer, and leukocytes were enriched by isolation over an 80/ plasmacytoid DC (pDC) in both the spleen and in the lungs. CD86 40% Percoll gradient (GE healthcare, Chicago, IL) after RBC lysis. was also higher on splenic cDC compared with splenic InfAPC Samples were resuspended in PBS containing 2% FBS and then throughout the infection, whereas CD80 distribution between treated with Fc block for 10 min at 4°C followed by surface staining APC subsets in the spleen varied over the course of the infection. for30minat4°C,exceptfor4-1BBL,CD70,OX40L,andGITRL, TNF family ligands were also preferentially expressed on InfAPC which were stained at room temperature for 30 min between Fc in the lung at day 3 postinfection (p.i.) with LCMV 13. block and the remaining surface staining. Samples from infected by guest on October 6, 2021 Previous results showed that IFN-I coordinately induces all mice were fixed in 4% paraformaldehyde or BD Cytofix Fixation four TNF family ligands, with peak expression at day 2 p.i. in the Buffer following surfacing staining. Data were acquired on a spleen. In this article, we show that there are two patterns of TNF Fortessa X20 or an LSR Fortessa with FACSDiva software. Data family ligand expression at the chronic phase of LCMV infection. analyses were performed using FlowJo v10. Gating strategies are CD70 is induced early during viral infection and then decreases to defined in Supplemental Figs. 1 and 2. at or below baseline within a few days and remains minimally expressed, as previously reported for GITRL (6, 11). In contrast, In vitro thioglycolate-elicited macrophage assays 4-1BBL and OX40L surface expression remain above baseline Mice were injected i.p. with 3% Brewers Thioglycolate medium through the chronic phase of LCMV 13 infection. In vivo, we show aged at room temperature for at least 3 mo.

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