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1.
Figure 2

Figure 2. LEAPS-loaded DCs target the site of infection.. From: Antigen-activated dendritic cells ameliorate influenza A infections.

107 CFSE-labeled nonpulsed DCs, JH-LEAPS–pulsed DCs (irrelevant LEAPS control), or combined-LEAPS–pulsed DCs were injected i.v. into naive mice (A) or influenza virus–infected mice (10 LD50, PR8 virus, i.n.) (B). The accumulation of CFSE-positive cells was measured in the lungs of mice 8, 24, and 48 hours after administration of the LEAPS-stimulated DCs. The data are representative FACS plots from each group (n = 5 mice/group).

Kobporn Boonnak, et al. J Clin Invest. 2013 July 1;123(7):2850-2861.
2.
Figure 4

Figure 4. LEAPS treatment is effective when administered at 24 or 48 hours after infection.. From: Antigen-activated dendritic cells ameliorate influenza A infections.

Mice infected with 10 LD50 of PR8 received an i.v. injection of NP-LEAPS– or JH-LEAPS–pulsed DCs injected at 24, 48, or 72 hours after infection. Percentage survival (A), percentage change in body weight (B), and virus titer in the lungs 4 days after infection are presented (C). In C, the mean titer from each group is represented as a solid bar, and the dashed line represents the low limit of detection.

Kobporn Boonnak, et al. J Clin Invest. 2013 July 1;123(7):2850-2861.
3.
Figure 3

Figure 3. M2e and NP peptides are critical determinants of efficacy of LEAPS treatment.. From: Antigen-activated dendritic cells ameliorate influenza A infections.

BALB/c mice were infected i.n. with 10 LD50 of PR8. Twenty-four hours after infection, mice received NP, HA1, HA2, or M2e-conjugated LEAPS pulsed DCs. LEAPS conjugated with a combination of these peptides was used as a positive control and JH-LEAPS was used as negative control. Percentage survival (A), percentage change in body weight (B), and virus titer in lungs harvested 4 days after infection are presented (C). In C, the mean titer from each group is represented as a solid bar and the dashed line represents the lower limit of detection.

Kobporn Boonnak, et al. J Clin Invest. 2013 July 1;123(7):2850-2861.
4.
Figure 8

Figure 8. NP-LEAPS DC treatment is effective in mice infected with an oseltamivir- resistant virus.. From: Antigen-activated dendritic cells ameliorate influenza A infections.

Mice infected with 106 TCID50 of A/Bethesda/NIH107-D31/2009 (oseltamivir-resistant pH1N1) were treated with oseltamivir phosphate (20 mg/kg/d) twice daily for 5 days starting 4 hours before infection. Oseltamivir was given alone or combined with NP-LEAPS pulsed DCs, which were given 24 hours after infection. The control group received sterile water by oral gavage twice daily for 5 days and 200 μl sterile PBS i.v. 24 hours after infection. Percentage of survival (A), percentage of changes in body weight (B), and virus titer in lungs (C) harvested 6 days after infection are presented.

Kobporn Boonnak, et al. J Clin Invest. 2013 July 1;123(7):2850-2861.
5.
Figure 1

Figure 1. Combined influenza-J-LEAPS–loaded DCs reduce morbidity and mortality in influenza virus–infected mice.. From: Antigen-activated dendritic cells ameliorate influenza A infections.

BALB/c mice were infected i.n. with 10 LD50 (A and B) or 1000 LD50 (C and D) of PR8. Twenty-four hours after infection, the mice were given an i.v. injection of PBS, nonpulsed DCs, or a combination of NP-, HA- and M2e-conjugated LEAPS–pulsed DCs. Virus titers were measured in the lungs harvested 4 days after infection. P = 0.005, Student’s t test for low-dose infection (A); P = 0.001, Student’s t test for high-dose infection (C). The mean titer from each group is represented as a solid bar. The dashed line represents the lower limit of detection. Percentage survival following infection was monitored for 14 days; P ≤ 0.001 for low-dose infection (B); P = 0.005 for high-dose infection (D), log-rank (Mantel-Cox) test.

Kobporn Boonnak, et al. J Clin Invest. 2013 July 1;123(7):2850-2861.
6.
Figure 5

Figure 5. LEAPS treatment results in decreased proinflammatory cytokines and increased Th1 cytokines in the lungs of PR8-infected mice (A). . From: Antigen-activated dendritic cells ameliorate influenza A infections.

On day 4 after infection, lung homogenates were analyzed for cytokine protein expression. Each data point represents the result from an individual mouse; horizontal bars indicate the mean. P values were determined by Student’s t test (***P < 0.001; **P < 0.01; *P < 0.05). Influenza-LEAPS elicit influenza-specific antibodies of the IgG2a subclass (B). Influenza-specific IgG1 and IgG2a subclass ELISA antibodies were determined in sera from PR8-infected mice collected on day 14 after infection. Infected mice were treated with DCs pulsed with NP-LEAPS, M2e-LEAPS, or combined-LEAPS influenza peptides. Control mice were infected i.n. with 100 TCID50 of PR8, and sera were collected on day 14 after infection. The error bars represent SEM for each group.

Kobporn Boonnak, et al. J Clin Invest. 2013 July 1;123(7):2850-2861.
7.
Figure 7

Figure 7. Depletion of CD8+ T cells reduces the efficacy of influenza-J-LEAPS treatment. . From: Antigen-activated dendritic cells ameliorate influenza A infections.

Schematic for depletion of CD8+ T cells in PR8-infected mice in combination with influenza-J-LEAPS treatment (A). FACS analysis of CD4 and CD8 expression on lymphocytes isolated from lymph nodes, lungs, spleen, and whole blood of mice treated with isotype or anti-CD8 mAbs i.p. 3 days and 1 day before and 2 days after influenza virus infection. The recorded numbers denote the percentages of CD4+ and CD8+ cells in the total lymphocyte population (B). Virus titers in the lungs were determined 4 days after infection (C). The mean titer from each group is represented as a solid bar and the dashed line represents the lower limit of detection.

Kobporn Boonnak, et al. J Clin Invest. 2013 July 1;123(7):2850-2861.
8.
Figure 6

Figure 6. NP-LEAPS–pulsed DCs elicit a rapid influenza-specific CD8+ T cell response in the lungs of PR8 virus–infected mice. . From: Antigen-activated dendritic cells ameliorate influenza A infections.

Mice were infected with 10 LD50 of PR8 and received NP-LEAPS– or JH-LEAPS–pulsed DCs as described in Figure 1. On day 4 after infection, lungs were harvested and homogenized and virus titers were determined (A), and isolated cells from lung tissues were stained with anti-CD3, anti-CD8, and NP147–155 pentamer. The percentage (B) and the number (C) of CD8+NP147–155 pentamer+ T cells were enumerated by flow cytometry. The percentage (D) and number (E) of influenza-specific CD8+ T cells in the lungs enumerated on days 3, 5, 8, or 11 after infection are presented, except the JH-LEAPS group (indicated with ++), in which the animals were sacrificed on day 9 after infection because they were moribund. The error bars represent SEM for each group, P < 0.05; Student’s t test. H&E staining was performed on lung sections from PR8 virus–infected mice that received JH-LEAPS DCs (middle column) or NP-LEAPS DCs (right column) 24 hours after infection. The lungs were harvested on day 4 after infection and mock-infected mice were used as control (left column) (F). Original magnification, ×400.

Kobporn Boonnak, et al. J Clin Invest. 2013 July 1;123(7):2850-2861.

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