1. Volume 47, Issue 5, Pages 943-958.e9 (November 2017)
Dichotomous Expression of TNF Superfamily Ligands on Antigen-Presenting Cells Controls Post-priming Anti-viral CD4+ T Cell Immunity 
Yu-Han Chang, Kuan Chung Wang, Kuan-Lun Chu, Derek L. Clouthier, Anh T. Tran, Miguel S. Torres Perez, Angela C. Zhou, Ali A. Abdul-Sater, Tania H. Watts 
Immunity 
Volume 47, Issue 5, Pages e9 (November 2017)
DOI: /j.immuni
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2. Immunity 2017 47, 943-958.e9DOI: (10.1016/j.immuni.2017.10.014)
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3. Figure 1
Antigen-Presenting Cell Subsets Show Dichotomous Expression of CD28 and TNFR Family Ligands
(A–C) Splenic APC subsets from D2 p.i. with LCMV13 were analyzed by flow cytometry using the gating strategies described in Figure S1A for (A) CD80, CD86, MHCII, and PD-L1 and (B) GITRL, 4-1BBL, OX40L, and CD70. Right: representative histograms. Abbreviations are as follows: InfDC, inflammatory DCs; InfMF, inflammatory macrophages; cDCs, classical DCs; and RPMF, red pulp macrophages. Left: summary plots reflect mean ± SEM of 6–9 mice pooled from 2–3 independent experiments.
(C) GITRLhi and CD80hi subsets from CD3−CD19−B220− splenocytes were analyzed by flow cytometry for MHCII, CD86, Ly6C, 4-1BBL, OX40L, and CD70. Data are displayed as mean ± SEM, from 6 mice pooled from 2 independent experiments.
(D) Mice were infected intranasally with influenza A/PR8 and draining LNs analyzed on D3 p.i. Gating on CD80hi or GITRLhi populations was conducted as in (C). Representative histograms are shown at left and summary data shown at right for 6 mice pooled from 2 independent experiments. dMFI refers to the MFI for the specific antibody stain minus the FMO control. MFI∗∗ reports the raw MFI for MHC II stain. Two-tailed, non-parametric paired t test was used for statistical analysis for all experiments.
See also Figure S1.
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4. Figure 2
GITRL Expression Kinetics In Vivo Suggests Its Regulation by IFN-I and IFN-I Induces GITRL Protein and Tnfsf18-Reporter Activity In Vitro
(A) Splenocytes were analyzed by flow cytometry on D0 to D21 post-LCMV13 infection for GITRL expression on the APC subsets defined in Figure S1A. Results show the mean ± SEM of 6 to 9 mice pooled from at least 2 independent experiments. Two-tailed, non-parametric paired t test was performed against D0 p.i.
(B) Total mRNA was extracted from splenocytes harvested at D0 to D21 p.i. and the indicated genes analyzed by qPCR. Expression is normalized to Gapdh and displayed as fold change relative to D0 p.i. Each symbol shows mean ± SEM pooled from 3 mice.
(C–F) Thioglycolate-elicited macrophages (TG MF) were harvested from mouse peritoneal cavity for in vitro culture.
(C) TG MF were treated with IFN-α4, -β, or -γ at 0–100 U/mL for 24 hr and analyzed for GITRL expression by flow cytometry with summary data and representative histograms shown.
(D) TG MF were pre-treated with 10 μg/mL of IFNAR blocking antibody or isotype control 1 hr prior to culturing with 0 or 100 U/mL of IFN-α4 or IFN-β for 24 hr, followed by flow cytometry analysis of GITRL expression.
(E) TG MF pre-treated in vitro with 25 U/mL of IFN-β for 24 hr were placed in fresh media with either 0 or 25 U/ml of IFN-β. GITRL expression was analyzed at 0, 24, and 48 hr following change of media.
(F) TG MF were infected in vitro with LCMV13-GFP at an MOI of 0.1 following pre-treatment with IFNAR blocking or isotype antibodies as in (D). Percent GFP+ and GFP dMFI of LCMV-GFP-infected cells at 24 hr p.i. are summarized in (F) at left with corresponding GITRL expression shown in (F) at right.
Data in (C)–(F) represent mean ± SEM of 3–6 independent cultures harvested from separate mice. Two-tailed, non-parametric paired t test was used for statistical analyses.
(G) Regulation of mouse GITRL transcription was determined using Cypridina/Gaussia-Dura dual luciferase assay in RAW264.7 cells. Top: Schematic of reporter constructs used with predicted IFN response elements indicated. Bottom: Response of WT and deleted construct to IFN-I and IFN-II. Results represent mean ± SEM of 3 independent experiments.
See also Figure S2.
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5. Figure 3
IFN-I Regulates TNF Family Induction during LCMV Clone 13 Infection In Vivo
Mice were injected i.p. with IFNAR blocking or isotype antibodies at D-1 and 0 prior to LCMV13 infection.
(A) GITRL, 4-1BBL, CD70, and OX40L surface expression was analyzed on splenic APC subsets by flow cytometry at D2 p.i.
(B and C) Splenic APC subsets were analyzed at D0 p.i. for IFNAR-1 expression (B) and at D1 p.i. for STAT1 Y701 phosphorylation (pSTAT1) (C) by flow cytometry. Representative histograms are shown next to summary plots.
Bars show mean ± SEM of 7 to 8 mice pooled from 2 independent experiments. Two-tailed, non-parametric unpaired (A) and paired (B, C) t tests were performed for statistical analyses. See also Figures S2 and S3.
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6. Figure 4
GITR-Dependent Phospho-Signaling Is Temporally Segregated from Initial T Cell Activation during LCMV13 Infection
CD45.2 wild-type C57BL/6 mice received a 1:1 mix of 106 each of Tnfrsf18+/+ and Tnfrsf18−/− CD45.1 SMARTA TCR-tg CD4+ T cells i.v. at D-1 p.i. as illustrated in (A). Tnfrsf18+/+ and Tnfrsf18−/− SMARTA TCR-tg CD4+ T cells were gated from splenocytes, as shown in (B), for analyses of percent and MFI of (C) CD69 and 4-1BB expression at 0 and 12 hr p.i. and (D) NF-κB p65 and pS6 at 0, 12, 24, 48, and 72 hr p.i. Data in (C) and (D) reflect mean ± SEM of 8 mice pooled from 2 independent experiments. Two-tailed, non-parametric paired t test was used for statistical analyses. See also Figure S4.
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7. Figure 5
Tnfrsf18+/+ and Tnfrsf18−/− CD4+ T Cells Show Differential Gene Expression during LCMV13 Infection
CD45.2+/+ wild-type C57BL/6 mice received 106 each of CD45.1+/+ Tnfrsf18+/+ and CD45.1+/− Tnfrsf18−/− SMARTA TCR-tg CD4+ T cells i.v. at D-1 p.i. At D3 p.i., Tnfrsf18+/+ and Tnfrsf18−/− SMARTA TCR-tg CD4+ T cells were subsequently sorted from purified CD4+ T cells pooled from 12 mice as illustrated in (A). RNA-sequencing results analyzed for differential gene expression as depicted in (B) with –log10(p value) versus log2(fold change relative to Tnfrsf18+/+). FDR threshold of 0.2 is marked by a horizontal dashed line and differential gene expression >1.2- or <−1.2-fold is indicated by red and blue, respectively. (C) Genes with differential expression of >1.5 or <−1.5-fold in (B) were categorized by functions. (B, C) Genes displayed were pre-filtered with RPKM > 0.1 and FDR < 0.2. (D) Surface expression of IL2Rα, IL-7R, OX40, HVEM, CX3CR1, and Ly6C was analyzed by flow cytometry with set-up identical to (A). Bars in (D) reflect mean ± SEM of 5–8 mice pooled from 2 independent experiments. Two-tailed, non-parametric paired t test was used for statistical analyses. (E) Analysis of Ly6C and CX3CR1 co-expression on SMARTA TCR-tg T cells from the experiment in (D), left representative flow cytometry plots, right summary of results for 8 mice pooled from 2 independent experiments. (F) Survival of cells from the experiments in (D). See also Figure S5.
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8. Figure 6
Deletion of Tnfsf18 Exon 2 in All Cells or Only in Myeloid Cells Has a Similar Effect on GITR-Dependent Signaling and on the Expansion of Tnfrsf18+/+ SMARTA as Compared to Tnfrsf18−/− SMARTA Cells Competing in the Same Mouse
Lyz2-Cre+/+ or Lyz2-Cre−/− × Tnfsf18exon2fl/fl littermates or CMV-Cre+/+ × Tnfsf18exon2fl/fl mice received 106 each of CD45.1+/+ Tnfrsf18+/+ and Tnfrsf18−/− SMARTA TCR-tg CD4+ T cells i.v., as illustrated in (A). Mice were infected 1 day later with LCMV13 and analyzed by flow cytometry (B) at day 3 for upregulation of pS6, CD25, or OX40 on Tnfrsf18+/+ or Tnfrsf18−/− SMARTA CD4+ T cells, (C) on day 8 for proportion and numbers of Tnfrsf18+/+ or Tnfrsf18−/− SMARTA CD4+ T cell populations, or (D) for frequency and number of IFN-γ production in Tnfrsf18+/+ or Tnfrsf18−/− SMARTA CD4+ T cell populations after peptide restimulation. Data in (B)–(D) represent 8 mice pooled from 2 experiments. Two-tailed, non-parametric paired t test was used for statistical analyses. See also Figure S6.
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9. Figure 7
Selective Deletion of Tnfsf18 Exon 2 in Myeloid Cells Reduces Endogenous Th1 CD4+ and LCMV-Specific CD8+ T Cell Responses and Increases Viral Load during LCMV13 Infection
Splenocytes from Tnfsf18exon2fl/fl littermates with either Lyz2-Cre+/+ or Lyz2-Cre−/− genotypes were analyzed by flow cytometry (A–H) or foci forming assay (I) at D8 or 21 p.i.
(A and B) Gating strategy for CD44hi T-bet+ Th1 population of CD4+ T cells (A), with frequency and number of cells recovered (B).
(C and D) GP61-80-tetramer+ CD4+ T cells (C) or IFN-γ+ CD4+ cells after GP61-80 restimulation (D).
(E and F) GP33-41, GP , or NP tetramer+ population of CD8+ T cells with representative flow cytometry histograms (E) along with summary plots (F).
(G) Frequency and number of IFN-γ-producing CD8+ T cells after restimulation with the indicated peptides.
(H) PD-1 expression on GP33-41+CD8+ T cells was analyzed.
(I) Viral titers.
Data (B–D, F–I) indicate the mean ± SEM of 7–8 mice per group pooled from 2 independent experiments. Two-tailed, non-parametric unpaired t test was used for statistical analyses. See also Figure S6.
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