Influenza infection induces weight loss, pulmonary inflammation, and OX40 expression by T cells. (A) Mice were infected with influenza, BAL removed 2, 4, 7, 11, and 15 d after infection, and total viable cells were enumerated (right axis). Weight loss was monitored daily and expressed as percent of original body weight (left axis). Results represent mean values ± SEM from four mice per group. BAL (B and C) and MLNs (D and E) were removed from influenza-infected mice and cells were stained with OX40-FITC and APC-CD4 (B and D) or PercP-CD8 (C and E). Results represent the mean percent (left) and total number (right) of OX40⁺ T cells ± SEM from five individual mice and is representative of three separate experiments.

OX40–Ig treatment prevents illness and T cell inflammation during influenza infection. (A) BALB/c mice (n = 4) were infected with influenza, treated with mouse IgG or OX40–Ig on alternate days, and weight loss was monitored and expressed as percent of original weight (P < 0.05 days 2–6). Illness was scored from 1–5 based on the degree of immobility, cachexia, and ruffled fur. The cumulative illness score was calculated by treatment group (B). (C) Total numbers of BAL CD4⁺ and CD8⁺ T cells 6 d after infection were determined by multiplying the percent of positive cells by flow cytometry with the total viable cells recovered from the BAL of IgG or OX40–Ig-treated mice. (D) In an identical experiment, BAL CD4⁺ and CD8⁺ T cell numbers were determined in mice treated with IgG, OX40–Ig, and OX40–mutIgG (gray symbols). Open symbols, OX40–Ig-treated mice; closed symbols, control IgG–treated mice. E and F show representative hematoxylin and eosin–stained lung sections 7 d after influenza infection in OX40–Ig- (E) or IgG- (F) treated mice. All experiments were repeated two to three times with five mice per group.

OX40–Ig reduces TNF and tetramer binding of CD8⁺ T cells. (A) Influenza-infected mice were treated with IgG or OX40–Ig and lung cells were stained for CD8 and intracellular TNF. Total numbers were calculated by multiplying total viable cells by the percent of CD8⁺/TNF⁺ cells. Mean percent (left) and total numbers (right) ± SEM from five mice per group are shown. Representative plots of lung CD8 versus TNF in lymphocytes 7 d after infection in IgG- (left) or OX40–Ig- (right) treated mice (B). (C and E) Lung cells were removed from IgG- or OX40–Ig-treated, influenza-infected mice 0, 2, 5, 7, and 10 d after infection and stained with anti–CD8-PercP, tetramer-APC, and anti–TNF-FITC. Total numbers were calculated by total viable cells × percent CD8⁺/Tetramer⁺ (C) or CD8⁺/Tetramer⁺/TNF⁺ (D). Results are mean values ± SEM of five mice per group. Representative plots of CD8 versus tetramer are shown in E in IgG- (left) or OX40–Ig- (right) treated mice 7 d after infection. All results are representative of at least two independent experiments containing five mice per group. Open symbols, OX40–Ig-treated mice; closed symbols, IgG-treated mice.

OX40–Ig reduces established illness and T cell proliferation, increases apoptosis, but does not inhibit viral clearance or recall responses. (A) Mice (n = 3) were infected with influenza virus on day 0 and treated with IgG or OX40–Ig on days 3, 4, and 5 after infection. Weight loss is expressed as percent of original weight. (B) Mice (n = 4) were infected with influenza and treated with either IgG or OX40–Ig. On days 2, 5, 7, and 11 after infection, lung tissue was homogenized and viral titre was quantified by plaque assay. (C–E) Mice (n = 4) were infected with influenza and treated with IgG or OX40–Ig as in B. 3 wk later mice were challenged with influenza and on days 2, 5, and 7 after infection, CD4⁺ (C), CD8⁺ (D), and CD8⁺ tetramer⁺ (E) T cells were enumerated in lung tissue. (F and G) Proliferating MLN CD4 and CD8 T cells (F, by BrdU incorporation) and apoptotic annexin V–stained lung cells (G) were assessed in control- or OX40–Ig-treated mice 4 and 7 d, respectively, after influenza infection. Closed symbols, IgG control; open symbols, OX40–Ig. Mean values ± SEM are shown and are representative of two separate experiments.