Fig 1.
DC-LM immunization induces robust memory CD8 T cell responses that mediate clearance following RSV infection.
Naive BALB/c mice were primed with mature DCs coated in M282 peptide and boosted 7 days later with recombinant LM expressing the M282 epitope. Control mice received mature DCs not coated in peptide and were inoculated with LM that did not express an RSV-derived epitope. (A) M282-tetramer+ CD8 T cell response was assessed in the PBL following DC-LM prime-boost immunization. (B) RSV titers at day 4 p.i. were determined via plaque assay in the lung. (C) Total CD8 and (D) M282-specific CD8 T cells were quantified in the lungs of immunized mice at days 0, 4, and 5 p.i. (E) Frequency of CD8 T cells in the lung producing either IFN-γ or IFN-γ and TNF after M282-peptide re-stimulation was determined at days 0, 4, and 5 p.i. Data are represented as mean ± SEM of two independent experiments (n = 8 mice). Groups were compared using Student’s t test for (A, B) and one-way ANOVA in (C-E), ** p<0.01, *** p<0.001.
Fig 2.
Memory CD8 T cells contribute to lethal pulmonary immunopathology upon RSV challenge.
Control- and M282-immunized mice were assessed for (A) survival, (B) weight loss, (C) Penh, and (D) MVb following RSV infection. (E) Lungs from naive, control, and M282 DC-LM-immunized mice were collected at day 5 p.i. and sections were stained for H&E. Representative images were captured at 200X magnification. Black arrows highlight a region of cellular debris and fibrin partially obstructing the alveolar duct. Red arrows highlight multifocal hyaline membranes. (F) Diffuse alveolar damage (DAD) scores were determined on day 5 following infection. Data are represented as mean ± SEM of two independent experiments (n = 7 naive mice, n = 8 mice for control and M282 groups). Groups were compared using Student’s t test for (A-D) and one-way ANOVA in (F), * p<0.05, ** p<0.01, *** p<0.001.
Fig 3.
M282-specific CD8 T cell memory protects against lethal heterologous IAV-M282 infection.
Control and M282-immunized mice were challenged with either RSV or a 5 LD50 dose of IAV-M282 and monitored daily for (A) survival, (B) weight loss, (C) Penh, and (D) MVb. (E) IAV titers in the lungs of IAV-M282-infected M282-immunized mice were determined at day 4 or 6 p.i. via plaque assay. Data are represented as mean ± SEM of two independent experiments (n = 12 mice for (A-D), n = 8–10 mice for (E)). Statistical comparisons were made using Student’s t test in (E) and one-way ANOVA for (A-D), */# p<0.05, **/## p<0.01, ***/### p<0.001. Asterisks represent statistical significance between M282: RSV and M282: IAV-M282 groups, while pound symbols indicate a difference between Control: IAV-M282 and M282: IAV-M282 groups.
Fig 4.
Airway CD8 T cells secrete excessive amounts of IFN-γ upon RSV infection in M282-immunized mice.
IFN-γ protein amount was determined in the (A) lung and (B) serum at 0, 2, and 4 days following RSV challenge of immunized mice. (C) DC-LM-immunized mice were treated with 250 μg BFA 6 hours prior to tissue collection. In vivo IFN-γ production was assessed for major leukocyte populations in the lung 2 days following either mock or RSV infection. Frequency of IFN-γ-producing CD8 T cells based upon (D) CD11a expression and (E) M282 epitope specificity 2 days after either mock or RSV infection. (F) Frequency of IFN-γ+ CD8 T cells was assessed in the lung, BAL, mLN, spleen, and PBL at day 2 p.i. Data are represented as mean ± SEM of two independent experiments (n = 6 for control and n = 8 for M282 in (A, B); n = 8 for (C-F)). Groups were compared using Student’s t test for (A-E) and one-way ANOVA in (F), * p<0.05, ** p<0.01, *** p<0.001.
Fig 5.
Neutralization of airway IFN-γ prevents lethal immunopathology associated with pre-existing CD8 T cell memory.
M282-immunized mice were treated with either control IgG or anti-IFN-γ antibody i.n. once during the time of RSV challenge. DC-LM-immunized mice were assessed daily for (A) survival, (B) weight loss, (C) Penh, and (D) MVb following infection. (E) RSV lung titers were determined at day 4 p.i. (F) TNF protein levels in the lung were quantified at day 2 p.i. Data are represented as mean ± SEM of two independent experiments (n = 11 for (A-D) and n = 8 for E-F)). Statistical analysis was performed using Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.