Conserved and Differential Features of TNF Superfamily Ligand Expression on APC Subsets over the Course of a Chronic Viral 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

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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 , , 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 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 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. Thioglycolate-elicited that the induction of 4-1BBL and OX40L at day 5 p.i. on InfAPC (TG) macrophages were harvested with 10% FBS–PBS at 4 d is partially blocked by pretreatment with IFN-gR–blocking postinjection and cultured in complete media (RPMI 1640, 10% Abs, suggesting that both type I and type II IFN contribute to FBS) supplemented with combinations of IFN-g,anti–IFNAR-1 expression of these TNF family ligands. We also found that LPS Ab, anti–IFN-gR Ab, IgG1 isotype control, or IgG2a isotype control induction of TNF family ligands on macrophages is substantially as indicated in the figures. mediated by IFN-I signaling, thereby highlighting the importance of IFNs and monocyte-derived APC in provision of TNF family In vivo IFN-gR blockade ligands to T cells. For in vivo IFN-gR blockade, 500 mgofanti–IFNG-gRAborIgG2a isotype were injected i.p. at day 1, 2, and 3 (total of 1.5 mg/mouse) after LCMV 13 infection for day 5 p.i. readout. MATERIALS AND METHODS Statistical analysis LCMV 13 Statistical analyses and graphs were generated using GraphPad LCMV 13 from Dr. M. Oldstone (Scripps Research Institute) was Prism v6. Paired or unpaired two-tailed, nonparametric Student t propagated on BHK cells and assayed by focus forming assay, as tests were applied in these graphs as detailed in their respective

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figure legends. *p , 0.05, **p , 0.01, ***p , 0.001 and ****p , findings at the day 2 time point (6). Although CD86 was higher on 0.0001 were applied. cDC compared with InfAPC throughout the course of infection, CD80 was more variable between subsets over time (Fig. 1B, lower panels). Thus, it is unlikely to be the level of activation that differs RESULTS between the APC subsets that explains their differential MHC II and TNF family ligand expression; rather, it is APC subset-specific Two patterns of TNF superfamily costimulatory ligand effects. expression on APC subsets in the spleen over the course of LCMV infection Expression of costimulatory ligands in the lung during Previous results showed differential expression of TNF and B7 LCMV infection family ligands on APC subsets in the spleen at day 2 of chronic Previous work has suggested that TNF family ligands can sustain LCMV infection (6). Here, using a similar 11-parameter flow T cell responsesin tissues such as the lung(18, 19). However, which cytometry panel with slight modifications (Abs listed in Table I; APC express TNF family ligands locally in tissues has not been Supplemental Fig. 1), we investigated the expression of TNF and thoroughly investigated. Our previous studies showed that GITRL

B7 family ligands as well as MHC II levels on APC over a 21-d expression in liver was similar to that in the spleen, with highest Downloaded from period after LCMV 13 infection. Our panel defined InfAPC as expression on InfAPC, albeit with slightly delayed kinetics (6). CD32 CD192 B2202 CD64+ FceR1+ as previously defined (13, 14). Here we extended our analysis to analyze the expression of the These APC include both a CD11c+ CD11b+ inflammatory DC subset four TNF superfamily ligands on APC subsets in a peripheral as well as a CD11clo CD11b+ inflammatory macrophage population, tissue, the lung, following LCMV 13 infection. For the lung, the which were pooled for the purpose of Fig. 1, as they showed similar gating strategy used was modified to also include interstitial + + results (data not shown). Within the same experiment, we macrophages, CD11b DC, CD103 DC, and eosinophils (see http://www.immunohorizons.org/ monitored costimulatory molecule expression on the CD11chi, Supplemental Fig. 2). Similar to our observations in spleen at MHC IIhi population, which includes DC1 and DC2 (8), as well as day2,atday3afterLCMV13infection,InfAPC,includingthe on the B220hiPDCA1hi cells, which are the pDC. Previous results inflammatory macrophages and inflammatory DC, showed the had shown that GITRL expression peaks at day 2 p.i. and then highest expression of TNF family ligands in the lung tissue, with declines to below baseline, remaining persistently low for the first lower expression on other APC subsets (Fig. 2A). 3 wk of LCMV 13 infection (6, 15) (reproduced schematically for We also analyzed TNF superfamily surface expression at day 8 comparison, Fig. 1A top left). Here we show that CD70 shows a p.i., a time point at which the shows many of the similar pattern as previously reported for GITRL, with peak features of chronic immune stimulation (20). We focused here on expression at day 2, and then declining to at or below baseline for 4-1BBL and OX40L, as GITRL and CD70 expression are minimal the remainder of the 3 wk of the experiment (Fig. 1A, top right). at this time point. At day 8 p.i. in the lung, 4-1BBL and OX40L also by guest on October 6, 2021 In contrast, 4-1BBL and OX40L, although also induced early, showed the highest expression InfAPC (Fig. 2B) Taken together, continue to be expressed above baseline over the 3 wk. 4-1BBL these data show that TNF superfamily ligands show preferential expression peaks early, at day 2–3, like CD70 and GITRL, whereas expressiononInfAPCinthelungatday3and8p.i. OX40L had a second wave of expression at around day 5 that is The above studies analyzed the different costimulatory larger than the first wave (Fig. 1A, lower panels). Throughout the molecules on APC subsets in separate staining panels. We next course of the infection, the expression of TNF family ligands was used a simplified panel that allowed us to analyze coexpression of consistently higher on InfAPC than on cDC or pDC. Thus, InfAPC different costimulatory ligands on the same APC simultaneously preferentially express TNF family ligands through to the chronic (Fig. 3). In a previous report, we showed that the GITRLhi and phase ofinfectionand show two patterns of expression, with CD70 CD80hi populations observed at day 2 after LCMV 13 and GITRL expressed transiently during the first few days of showed dichotomous expression of TNF and B7 family ligands. infection, whereas 4-1BBL and OX40L show more persistent Here we extend these results to the lung. At day 3 p.i., we saw a expression. clear dichotomy between CD80hi and GITRLhi APC, with the We also monitored MHC II, PD-L1, CD80, and CD86 on GITRLhi APC showing highest expression of Ly6C and 4-1BBL InfAPC and cDC over the 3 wk. MHC II was consistently highest and lower expression of MHC II. However, in contrast to previous on cDC throughout the time course examined (Fig. 1B, top left). results in the spleen, CD86 was actually higher on the GITRLhi InfAPC and cDC had similar levels of PD-L1 at days 1–3, but cDC cells rather than the CD80hi cells in the lung (Fig. 3). had higher levels of surface PD-L1 than InfAPC at days 6–9of infection, although this difference was lost and slightly reversed by Type II IFN contributes in part to induction of 4-1BBL and day 21 (Fig. 1B, top right). It is possible that the PD-L1hi DC OX40L during chronic viral infection population includes the recently described regulatory DC IFN-IisresponsibleforthecoordinateinductionofGITRL, population that accumulates at the chronic stage of LCMV 4-1BBL, CD70 and OX40L during the first few days of chronic infection (16, 17). At day 1 p.i, CD80 surface expression was higher LCMV infection (6). However, GITRL surface levels are reduced on InfAPC compared with cDC, whereas at day 2 p.i. the reverse to below baseline by about day 6 of infection (6), similar to the was true, as shown in the inset histograms, confirming previous results reported in this article for CD70 (Fig. 1A). We attribute the

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FIGURE 1. Expression of TNF superfamily ligands, B7 family ligands, and MHC II on splenic APC over the course of LCMV 13 infection. Splenocytes were analyzed by flow cytometry on day (D) 0 to D21 after LCMV 13 infection using the gating strategy shown in Supplemental Fig. 1 for (A) CD70, OX40L, and 4-1BBL on cDCs, InfAPC, and pDCs, with a schematic of previous data for GITRL (6) shown top left for comparison, and (B) MHC II, PD-L1, CD80, and CD86 on cDCs and InfAPC. Results show the mean 6 SEM of six to nine mice pooled from at least two independent experiments. Two-tailed, nonparametric unpaired t test was used for statistical analysis between cDC and InfAPC for all experiments. dMFI refers to the MFI for the specific Ab stain minus the FMO control. The inset histograms show representative flow cytometry data for the cDC and InfAPC at day 2 p.i. *p , 0.05, **p , 0.01.

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FIGURE 2. Inflammatory APC show highest expression of TNF superfamily ligands in the lung after LCMV infection. Lung APC subsets from (A) day (D) 3 p.i. and (B) D8 p.i. with LCMV 13 were analyzed for TNF superfamily ligand expression by flow cytometry using the gating strategy described in Supplemental Fig. 2. Left, Summary plots. Right, Representative histograms color coded to match the summary plots at left. Data represent mean 6 SEM with six mice pooled from two independent experiments. Two-tailed, nonparametric unpaired t test was used for statistical analysis for all experiments. dMFI refers to the MFI for the specific Ab stain minus the FMO control. *p , 0.05, **p , 0.01. ns, not significant. decrease in GITRL levels to several factors, including infection of was only induced in response to IFN-I but not IFN-g on isolated a large proportion of APC by day 6 of infection, resulting in macrophages, perhaps explaining the lack of sustained GITRL suppression of the IFN response (6, 21). A candidate for another expression as compared with OX40L and 4-1BBL (6). To test the cytokine that might contribute to TNF ligand induction is IFN-g, role of IFN-g in induction of the other three TNF family ligands, as within a few days of infection, IFN-g–producing T cells start to we analyzed TG peritoneal macrophages treated ex vivo with accumulate. Moreover, previous results had shown that GITRL increasing doses of IFN-g. TG macrophages were chosen for these

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FIGURE 3. Dichotomous expression of B7 and TNF superfamily ligands in the lung after LCMV 13 viral infection. Lung APC subsets from day 3 p.i. with LCMV 13 were analyzed by flow cytometry where GITRLhi and CD80hi subsets from CD32 CD192 B2202 http://www.immunohorizons.org/ single-cell suspension were analyzed for MHC II, CD86, Ly6C, and 4-1BBL. Data are displayed as mean 6 SEM, from six mice pooled from two independent experiments. Representative histograms are shown at the top and summary data shown at the bottom. dMFI refers to the MFI for the specific Ab stain minus the FMO control. Two-tailed, nonparametric unpaired t test was used for statistical analysis for all experiments. **p , 0.01. in vitro assays, as there are very few inflammatory APC in the Induction of TNF family ligands on macrophages by LPS is spleen of the mice prior to infection (see Supplemental Fig. 1), and largely dependent on IFN-I isolated InfAPC did not survive well in culture (data not shown). There have been many reports that TNF superfamily members are On TG macrophages, 4-1BBL expression was most responsive to induced by TLR signals, such as LPS stimulation (22); however, whether this is directly due to signaling downstream of TLRs or IFN-g, showing a dose-dependent increase in expression. There by guest on October 6, 2021 was also a small, statistically significant increase in both CD70 and due to signaling induced by downstream cytokines was not clear. OX40L expression on thioglycolate macrophages in the presence Many innate immune stimulatory pathways, such as TLR4, induce of IFN-g; however, this expression showed minimal dose de- IFN-I (23). To investigate whether LPS induction of TNF family pendence (Fig. 4A). There was also reduced induction of all three ligands was also mediated through IFN-I receptor signaling, we TNF family ligands in the presence of blocking Abs to IFN-gR treated TG macrophages with LPS in the presence or absence of (Fig. 4B). However, thioglycolate macrophages are activated by the anti-IFNAR1 Abs. LPS showed a dose-dependent induction of all four TNF family ligands (Fig. 5A). The induction of GITRL, CD70, induction procedure and show expression of TNF family ligands at and OX40L was substantially blocked by anti-IFNAR1, whereas baseline, which is also reduced by anti–IFN-gR, making these induction of 4-1BBL was only partially blocked (Fig. 5B). These studies somewhat difficult to interpret. studies show that induction of TNF superfamily ligands by LPS is Therefore, to test whether IFN-g playedaroleinexpressionof largely mediated by IFN-I signaling for GITRL, OX40L, and CD70, OX40L and 4-1BBL in vivo, we treated mice with Abs to IFN-gRat whereas 4-1BBL induction by LPS shows an IFNAR1-dependent day 1, 2, and 3 p.i. and monitored 4-1BBL and OX40L expression on and an IFNAR1-independent component. InfAPC and cDC on day 5. The results show a small but significant decrease in expression of 4-1BBL and OX40L on InfAPC after anti–IFN-gR treatment in vivo, showing that the expression of DISCUSSION these TNF family ligands is partially mediated by IFN-g (Fig. 4C). It should be noted that at these time points, based on our previous The TNF superfamily ligands GITRL, 4-1BBL, OX40L, and CD70 findings (6), there would also be significant induction of 4-1BBL are important costimulatory ligands for T cell survival with and OX40L by IFN-I, so the partial effect of anti–IFN-gRisnot numerous studies showing their importance in sustaining T cell surprising. GITRL and CD70 were not monitored at this time responses (4, 5, 22, 24). Moreover, agonistic Abs to each of the point, as their expression is minimal at day 5. These findings show TNFRs in this pathway are in clinical trials for (25) (see also that IFN-g contributes partially to the expression of 4-1BBL and www.clinicaltrials.gov). Thus, there is much interest in under- OX40L in vivo. standing the regulation of TNF family ligands in vivo. At the early

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FIGURE 4. IFN-g induces TNF family ligand protein expression in vitro and in vivo. (A and B) TG macrophages (TG MF) were harvested from mouse peritoneal cavity for in vitro culture. (A) TG MF were treated with IFN-g at 0–100 U/ml for 24 h and analyzed for 4-1BBL, CD70, and OX40L expression by flow cytometry with summary data and representative histograms shown. Results represent mean 6 SEM of six independent cultures harvested from separate mice and two independent experiments. Two-tailed, non- parametric unpaired t test were performed for statistical analyses. *p # 0.05, **p # 0.01. (B) TG MF were pretreated with 200 mg/ml of IFN-gR blocking Ab or isotype control 3 h prior to culturing with 0 or 50 U/ml of IFN-g followed by flow cytometry analysis for TNF family ligand expression. Results represent mean 6 SEM of four experiments using independent cultures of TG MF harvested from three to four separate mice per experiment. (C) Mice were injected i.p. with IFN-gR blocking or isotype control Abs at day (D) 1, 2, and 3 p.i. (500 mg each injection). Splenic APC subsets were analyzed by flow cytometry at D5 p.i. using the gating strategy defined in Supplemental Fig. 1 for 4-1BBL surface expression. Bars show mean 6 SEM of eight mice pooled from two independent experiments. Two-tailed, nonparametric unpaired t test were performed for statistical analyses (B and C). *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant.

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FIGURE 5. LPS induces TNF superfamily ligand protein expression on thioglycolate macrophages via IFN-I. (A and B). TG macrophages (TG MF) were harvested from mouse peritoneal cavity for in vitro culture. (A) TG MF were treated with LPS at 0–1000 pg/ml for 24 h and analyzed for GITRL, 4-1BBL, CD70, and OX40L expression by flow cytometry with summary data and representative histograms shown. (B) TG MF were pretreated with 50 mg/ml of IFNAR blocking Ab or isotype control, 1 h prior to culturing with 0 or 1000 pg/ml of LPS followed by flow cytometry analysis of the ligands’ expression. Results represent mean 6 SEM of five to eight independent cultures harvested from separate mice and two independent experiments. Two-tailed, nonparametric unpaired t test was used for statistical analyses (A and B). *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant.

stages of chronic infection, these four TNF family ligands are lung APC, in the lung, the GITRLhi 4-1BBLhi Ly6C hi InfAPC coordinately induced in the spleen by IFN-I preferentially on subset, rather than the CD80hi subset of APC, had the highest InfAPC compared with classical DC or pDC (6). Here we have expression of CD86. extended the analysis of TNF superfamily surface expression to We observed two patterns of TNF superfamily ligand expres- show that the preferential expression of TNF family ligands on sion during chronic LCMV 13 infection (Fig. 1). CD70 expression monocyte-derived APC extends through to the chronic stage of on APC, as previously reported for GITRL, is restricted to the first infection and is also observed in lung. Thus, the finding that cDC few days of infection, after which it returns to at or slightly below have highest expression of MHC II, whereas monocyte-derived preinfection levels. For GITRL, the surface expression follows APC have highest levels of TNF family ligands, is generalizable shortly after the induction of GITRL mRNA and IFN-I mediated from the early to chronic stage of infection and also in the tissues. induction of GITRL was shown to be transcriptionally medi- Although CD86 was generally higher on cDC than InfAPC in the ated (6). Moreover, CD70 and GITRL have been noted as IFN- spleen, CD80 distribution between splenic APC subsets fluctuated stimulated genes in the Interferome database (www.interferome. over the course of the infection. Moreover, although there was org), suggesting that this regulation is likely transcriptional. For 4- dichotomous expression of CD80 versus TNF family ligands on 1BBLandOX40L,weobservedadifferent pattern of expression.

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TABLE I. Abs and reagents Reagent Source Catalog Identifier Abs Anti-CD16/32 (93) eBioscience 14-0161-82 Anti-B220 (RA3-6B2)–PerCP-eF710 eBioscience 47-0452-82 Anti-FceR1 (MAR-1)–PE-Cy7 eBioscience 25-5898-82 Anti-NK1.1 (PK136)–PE, allophycocyanin-eF80 eBioscience 47-5941-82 47-5941-82 Anti–PDCA-1 (eBio927)–allophycocyanin, FITC eBioscience 17-3171-82 11-3172-82 Anti-CD11c (N418)–AF700, allophycocyanin eBioscience 56-0114-82 17-0114-82 Anti-CD11b (M1/70)–FITC eBioscience 11-0118-42 Anti-F4/80 (BM8)–allophycocyanin-eF780 eBioscience 47-4801-82 Anti–MHC II (I-A/I-E M5/114.15.2)–eF450, PE eBioscience 48-5321-82 12-5321-82 Anti-CD8a (53-6.7)–allophycocyanin eBioscience 17-0081-82 Downloaded from Anti-CD103 (2E7)–allophycocyanin eBioscience 17-1031-82 Anti-CD80 (16-10A1)–PerCP-eF710, Biotin eBioscience 46-0809-42 13-0801-82 Anti–PD-L1 (MIH5)–PerCP-eF710 eBioscience 46-5983-42 Anti-CD86 (GL-1)–allophycocyanin, FITC, eBioscience 17-0862-82 Biotin 11-0862-82 13-0862-82 http://www.immunohorizons.org/ Anti–Ly-6C (HK1.4)–allophycocyanin-eF780, eBioscience 47-5932-82 PE-Cy7 25-5932-82 Anti-CD19 (eBio1D3)–Biotin eBioscience 13-0193-82 Mouse IgG2a (eBM2a)–Biotin eBioscience 13-4724-83 Anti-CD3 (17A2)–BV605 BioLegend 100237 Anti-CD3 (2C11)–Biotin This article N/A Anti-CD19 (6D5)–BV605, Biotin BioLegend 115539 Anti-B220 (RA3-6B2)–BV605 BioLegend 103243 Anti-CD64 (X54-5/7.1)–BV711, allophycocyanin BioLegend 139311 139306 Anti-CD70 (FR70)–Biotin BioLegend 104603 Anti-OX40L (RM134L)–Biotin BioLegend 108803 by guest on October 6, 2021 Anti-GITRL (MIH44)–PE BD Biosciences 563541 Anti-Mouse 4-1BBL (19H3)–Biotin Purified from hybridoma provided by N/A Dr. R. Miller, Emory University, and labeled using molecular probes N-hydroxy succinimidyl biotin Anti-IFNAR1 Ab (MAR1-5A3), unconjugated/ Bio X Cell BE0241 functional grade Anti–IFN-gRAb (GR-20), unconjugated/ Bio X Cell BE0029 functional grade Mouse IgG1 isotype, unconjugated Bio X Cell BE0083 Rat IgG2a (2A3) isotype, unconjugated Bio X Cell BE0089

Chemicals, peptides, and recombinant proteins Fixable viability dye eF506 eBioscience 65-0866-14 Streptavidin-allophycocyanin (flow cytometry) eBioscience 17-4317-82 Streptavidin-PE (flow cytometry) eBioscience 12-4317-87 Murine IFN-b PBL Assay Science 12405-1 Murine IFN-g PeproTech 315-05 Brewer Thioglycollate Sigma-Aldrich B2551-500G BD Cytofix Fixation Buffer BD Biosciences 554655 Collagenase IV Invitrogen 17104019 Percoll GE healthcare 17089101 N/A, not applicable.

OX40L shows two waves of expression early on, peaking at day 2 cells (26), which would include the InfAPC. Although the initial and 5 (Fig. 1). This biphasic pattern of expression during the first induction of 4-1BBL and OX40L occurs coordinately with that of few weeks of LCMV 13 infection was previously reported on F4/80+ CD70 and GITRL and is IFN-I dependent (6), 4-1BBL and OX40L

https://doi.org/10.4049/immunohorizons.1800047 416 COSTIMULATORY LIGAND EXPRESSION DURING CHRONIC INFECTION ImmunoHorizons continue to showexpressionabovebaseline through to the chronic important regulator of signal 4 (TNF superfamily costimulation on stage of infection. It is possible that this second wave of expression T cells) than previously realized. There is currently much interest on InfAPC in vivo is mediated in part by IFN-g (Fig.4C),as in use of IFN-I–inducing agonists such as the TLR7/8 agonist and induction of 4-1BBL and OX40L at day 5 was partially blocked by STING agonists into tumors for inducing T cell responses as anti–IFN-gR. The lack of a complete block of 4-1BBL and OX40L cancer therapy (32, 33). Thus, it is possible that TNF family ligands by anti–IFN-gR at day 5 (Fig. 4C) is likely due to IFN-I, which is on monocyte-derived APC contribute to the efficacy of such also active in inducing TNF family ligands during this time frame therapies. (6). We previously showed that the majority of APC are infected with LCMV by day 6 of infection and that LCMV-infected cells DISCLOSURES have reduced pStat1 signaling compared with bystander cells, suggesting that the high level of infection of APC by the chronic The authors have no financial conflicts of interest. stage of infection would prevent further induction of TNF family ligands (6). It should be noted, however, that induction of TNF family ligands does not correlate with pStat1 levels, as pDC express ACKNOWLEDGMENTS

high levels of pStat1 1 d p.i. but express minimal TNF family ligands Downloaded from (6). Thus, stat1-dependent IFN-I and -II signaling does not fully We thank Dionne White and Joanna Warzyszynska for assistance with explain TNF family ligand expression at the chronic stage of flow cytometry, Birinder Ghumman for technical assistance, and Yu-Han infection, and it is likely that pStat1 independent signals and/or Chang for critical reading of the manuscript. other cytokines contribute to TNF family ligand induction at the chronic phase of infection. Indeed, we showed that LPS-induced REFERENCES

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Supplemental Figure 1. Multiparameter flow cytometry gating strategy to delineate splenic APC subsets during LCMV 13 infection. Representative example of gating strategy for plasmacytoid DCs (pDC), classical DC (cDC), and inflammatory APC (Inf APC), including both inflammatory DCs and inflammatory macrophages are shown. In this example, a single cell suspension of splenocytes was prepared from C57BL/6NCrl female mice (6-10 weeks old) day 2 post LCMV 13 infection. Data were collected with Fortessa X20 with FACSDiva software. Data analyses were performed using FlowJo v10.

Supplemental Figure 2. Multiparameter flow cytometry gating strategy to delineate lung APC subsets during LCMV 13 infection. Demonstration of gating strategy for eosinophil, interstitial macrophages, classical DCs (cDC), including CD103+ DC and CD11b+ DC, and inflammatory APCs (Inf APC), including inflammatory DCs and inflammatory macrophages. In this representative example, a single cell suspension of lung was prepared from C57BL/6NCrl female mice (6-10 weeks old) day 3 post LCMV 13 infection. Data were collected with Fortessa X20 with FACSDiva software. Data analyses were performed using FlowJo v10.