Volume 41, Issue 4, Pages 646-656 (October 2014) TRAIL+ NK Cells Control CD4+ T Cell Responses during Chronic Viral Infection to Limit Autoimmunity  Iona S. Schuster, Matthew E. Wikstrom, Geraldine Brizard, Jerome D. Coudert, Marie J. Estcourt, Mitali Manzur, Lorraine A. O’Reilly, Mark J. Smyth, Joseph A. Trapani, Geoffrey R. Hill, Christopher E. Andoniou, Mariapia A. Degli-Esposti  Immunity  Volume 41, Issue 4, Pages 646-656 (October 2014) DOI: 10.1016/j.immuni.2014.09.013 Copyright © 2014 Elsevier Inc. Terms and Conditions

Immunity 2014 41, 646-656DOI: (10.1016/j.immuni.2014.09.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 TRAIL+ NK Cells Accumulate in the SGs during Chronic MCMV Infection (A) NK cells were enumerated in the SGs after infection with MCMV-K181 (5 × 103 PFU/mouse). Data are pooled from four independent experiments and plotted as mean ± SEM (n = 16/time point). (B) Expression of various cell surface markers on NK cells isolated from the SGs of MCMV-infected mice is shown. The histograms depict NK cells from uninfected mice (gray filled histograms) and MCMV-infected mice at day 18 pi (open black line histograms). The data are representative of a minimum of two independent experiments (n = 3–4 mice/experiment). (C) Expression of TRAIL on NK cells present in the SGs after MCMV infection was measured by flow cytometry. The data are representative of four independent experiments (n = 4 mice/experiment). (D) Absolute numbers of TRAIL+ NK cells present in the SGs of MCMV-infected mice. Data are pooled from four independent experiments and plotted as mean ± SEM (d0 n = 8, d10 n = 16, d18 n = 16). (E and F) Number of NK cells (E) and number of TRAIL+ NK cells (F) in the liver at various times pi. Data are pooled from at least two independent experiments with three to four mice each and plotted as mean ± SEM (d0 n = 11, d10 n = 12, d18 n = 16, d25 n = 8). ∗p < 0.05, ∗∗∗p < 0.001. p values for (A), (D), and (E) were obtained using a two-tailed Mann-Whitney test. See also Figure S1. Immunity 2014 41, 646-656DOI: (10.1016/j.immuni.2014.09.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 TRAIL Limits the Control of Chronic MCMV Infection in SGs (A) Viral loads in the SGs of BALB/c (WT) and BALB.Trail–/– (Trail–/–) mice infected with 104 PFU of MCMV-K181. SGs were collected at the indicated times and viral titers determined by plaque assay. Data are pooled from at least two independent experiments with at least three mice/group and plotted as mean ± SEM (d4 n = 6, d10 n = 9, d18 n = 9, d25 n = 13, d32 n = 7 for both WT and Trail–/–). (B) Viral loads in the SGs of BALB/c (WT) and BALB.Dr5–/– (Dr5–/–) mice infected with 104 PFU of MCMV-K181. Data are pooled from at least two independent experiments with at least three mice/group and plotted as mean ± SEM (d4, WT n = 8 and Dr5–/– n = 7; d10, WT n = 8 and Dr5–/– n = 6; d18, d25, and d32, WT n = 12 and Dr5–/– n = 10). ∗∗p < 0.01, ∗∗∗p < 0.001. p values were obtained using a two-tailed Mann-Whitney test. See also Figure S2. Immunity 2014 41, 646-656DOI: (10.1016/j.immuni.2014.09.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 TRAIL+ NK Cells Limit CD4+ T Cell Numbers in the SGs (A) Immunofluorescence staining (×20) of SGs from WT mice at day 18 after MCMV infection. αNKp46 (red) and αCD4 (green) were used to stain NK and CD4+ T cells, respectively. NK cells per field were enumerated, and the number of NK cells in contact with CD4+ T cells determined. Data from 11 representative fields from 3 individual mice are plotted as mean ± SEM. Data are representative of 3 independent experiments, each with at least 3 mice. (B) Expression of DR5 on CD4+ T cells harvested from the SGs at various times after MCMV infection. Data are representative of four independent experiments with at least three mice/time point. (C) Number of CD4+ T cells in the SGs of WT and Trail–/– mice at various times after MCMV infection. Data are pooled from at least two independent experiments with at least three mice/group and plotted as mean ± SEM (n = 7 or greater for both WT and Trail–/–). (D) Number of DR5+CD4+ T cells in the SGs of WT and Trail–/– mice at various times after MCMV infection. Data are pooled from two independent experiments with at least three mice/group and plotted as mean ± SEM (n = 7–8 per time point). (E) Expression of DR5 and CD44 on CD4+ T cells harvested from the SGs of WT mice at day 18 postinfection. Top panel shows a fluorescence minus one (FMO) control. The data are representative of two independent experiments with at least three mice. (F) Expression of DR5 and CD107a on CD4+ T cells harvested from the SGs of WT mice at day 18 postinfection. The top panel shows a FACS plot from an uninfected mouse. The data are representative of two independent experiments with at least three mice per group (n = 7). (G) Number of CD4+ T cells expressing DR5 and CD44hi in the SGs of WT and Trail–/– mice at day 18 pi. Data are plotted as mean ± SEM (n = 3 per group). (H) Number of CD4+ T cells expressing DR5 and CD107a in the SGs of WT and Trail–/– mice at day 18 pi. Data are pooled as mean ± SEM (n = 7–8 per group). ∗p < 0.05 obtained using a two-tailed Mann-Whitney test in (C), (D), and (H). ∗p < 0.05 obtained using a two-tailed t test in (G). Immunity 2014 41, 646-656DOI: (10.1016/j.immuni.2014.09.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 NKG2D Is Expressed on TRAIL+ NK Cells and NKG2DL on DR5+CD4+ T Cells (A) Frequency of SG NK cells expressing NKG2D after MCMV infection was measured by flow cytometry. The histogram depicts NKG2D expression on TRAIL+ NK cells at day 18 pi (open black line) versus a FMO control for NKG2D (gray filled histogram). (B) The percentages of TRAIL+ and TRAIL− NK cells expressing high amounts of NKG2D are shown. Data are pooled from two independent experiments (n = 8) and plotted as mean ± SEM. (C) Expression of NKG2D ligands on CD4+ and CD8+ T cells is shown in representative FACS plots. Analysis was performed on SG lymphocytes isolated at day 18 post-MCMV infection, using a mouse NKG2D-Fc fusion protein. (D) The percentages of CD4+ and CD8+ T cells expressing NKG2D ligands (NKG2DL) are shown. Data are plotted as mean ± SEM from one experiment (n = 3) and are representative of results from three independent experiments. (E) The expression of NKG2DL and CD44 on SG CD4+ T cells is shown together with the frequency of cells expressing DR5 within the NKG2DL-positive and NKG2DL-negative fractions of the CD44hiCD4+ T cell pool. Cells were isolated from the SGs of mice infected with MCMV (day 18). Immunity 2014 41, 646-656DOI: (10.1016/j.immuni.2014.09.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 TRAIL+ NK Cells Limit CD4+ T Cell Numbers and Affect Chronic Viral Loads (A) Viral loads in the SGs of MCMV-infected mice at day 25 pi. Trail–/– mice were depleted of CD4+ (500 μg GK1.5 mAb at day 9, followed by 250 μg at days 11, 16, and 20) or CD8+ (200 μg 53.5.8 mAb at day 9, followed by 100 μg at days 11, 16, and 20) T cells. ∗p < 0.05, ns = not significant; obtained using a one-way ANOVA with Tukey’s multiple comparison test. (B–D) Viral loads (B), frequency of DR5+CD4+ T cells (C), and number of DR5+CD4+ T cells (D) in the SGs of MCMV-infected mice at day 25 pi. WT mice (BALB.B6-CT6: H-2d, Ly49Hneg, NK1.1pos) were infected with MCMV-K181 and NK cells depleted with 200 μg PK136 on days 9, 13, 17, and 22 pi. Data are pooled from two independent experiments for viral loads (B, n ≥ 8) and from three independent experiments for enumeration of DR5+CD4+ T cells (C, D, n = 12). (E) WT or Trail–/– mice that received 3–3.5 × 106 naive WT or Trail–/– NK cells i.v. on day 4 pi were sacrificed on day 25 pi, together with control MCMV-infected WT and Trail–/– mice, and viral titers were assessed by plaque assay. WT n = 11, WT + WT NK cells n = 13, Trail–/– n = 8, Trail–/–+ WT NK cells n = 16, Trail–/–+ Trail–/– NK cells n = 5. (F) Trail–/– mice that received 3–3.5 × 106 naive WT or Trail–/– NK cells i.v. on day 4 pi were sacrificed on day 25 pi and the number of CD4+DR5+ T cells in the SGs enumerated (n = 3). Data are plotted as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, ns = not significant; p values were obtained using a one-tailed Mann-Whitney test (B–E) or a one-tailed t test (F). Immunity 2014 41, 646-656DOI: (10.1016/j.immuni.2014.09.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Autoimmunity after MCMV Infection Develops More Rapidly in the Absence of TRAIL Expression (A) IgM autoantibodies reacting against SG homogenates from day 40 MCMV-infected WT and Trail–/– mice. Data are pooled from two independent experiments (n = 8). (B) SGs from WT and Trail–/– mice at day 120 after MCMV infection were examined histologically. Black arrowheads denote the aggregates (scale bars represent 200 μm, 20× magnification). Images from H&E-stained sections are representative of results obtained in two independent experiments with three to five mice/group. (C) Anti-SSA antibody concentrations (IgG+IgA+IgM) in sera of naive and MCMV-infected WT and Trail–/– mice on days 40, 60, and 80 pi was assessed by ELISA. Data are pooled from up to four independent experiments (d0 n = 4, d40 n = 12, d60 n = 18, and d80 n = 9 for each strain) and plotted as mean ± SEM. (D) Saliva flow in WT and Trail–/– mice at day 80 after MCMV infection. Data are plotted as mean ± SEM (n = 6/group). ∗∗p < 0.01 was obtained using a one-tailed Mann-Whitney test. For (A) and (D), ∗p < 0.05, obtained using a one-tailed unpaired t test with Welch’s correction, since the data had a normal distribution. (E) Saliva flow at 72 days after MCMV infection in control and mice depleted of NK cells with 200 μg PK136 on days 9, 13, 17, and 22 pi. Data are plotted as mean ± SEM (n = 3/group). ∗p < 0.05, obtained using a one-tailed t test. (F) Tear production at 71 days after MCMV infection in control and mice depleted of NK cells with 200 μg PK136 on days 9, 13, 17, and 22 pi. Data are plotted as mean ± SEM (n = 4/group). ∗∗∗p < 0.001 was obtained using a one-tailed t test. Immunity 2014 41, 646-656DOI: (10.1016/j.immuni.2014.09.013) Copyright © 2014 Elsevier Inc. Terms and Conditions