Figure 1.
Viral titer kinetics in RSV-infected adult and neonatal mice.
(A) Viral titers in the lungs of mice at days 1-8 following infection with 2×106 PFU of RSV. Error bars represent the SEM. (B) Viral titers in the lungs of adult and neonatal mice at day 4after infection with 1×107 PFU of RSV. Horizontal bars represent the mean values. Data are representative of experiments performed twice with 4-8 mice/group.
Figure 2.
RSV epitope-specific CD8+ T cell responses during acute infection and memory.
(A) Tetramer-positive cells specific for KdM282-90 and DbM187-195 were measured in the lung at days 5-14 following infection of adult mice. Percentages of CD3+CD8+ cells staining with the indicated tetramers are shown. (B) Epitope-specific CD8+ T cell responses to KdM282-90 and DbM187-195 measured in the lung at 5-14 days post-infection in neonatal CB6F1 mice. (C) Epitope response ratios (KdM282-90/DbM187-195) measured from lung tissue at days 5-14 post-infection of adult and neonatal mice (D) Epitope-specific memory CD8+ T cell responses in the spleens of mice infected 72 days earlier as adults or neonates. Data are representative of 2-3 independent experiments with 4-8 mice/group. Error bars represent the SEM.
Figure 3.
Epitope-specific CD8+ T cell cytokine production and functional avidities in RSV-infected adult and neonatal mice.
(A) Frequency and cytokine production by CD8+ T cells specific for KdM282-90 and DbM187-195 in the lungs of RSV-infected adults and neonates. Parallel samples were stained with tetramer and incubated with the M282-90 or M187-195 peptide for 5 hours in the presence of the transport inhibitor monensin to measure intracellular cytokine staining by flow cytometry. Tetramer frequencies are indicated by the blue and red bars for the KdM282-90 and DbM187-195 CD8+ T cell populations, respectively. (B) Functional avidities of KdM282-90 and DbM187-195 –specific IFN-γ-producing CD8+ T cells in the lungs of infected adults and neonates. All data were normalized to 100% production at 10−6M of each peptide. (C) Functional avidities of KdM282-90 and DbM187-195 specific IFN-γ-producing CD8+ T cells in the spleens of infected adults and neonates. Data represent results from 3-4 independent experiments with 4-7 mice/group. Error bars represent the SEM.
Figure 4.
Epitope-specific CD8+ T cell responses following infection with rAd5-MM2.
(A) KdM282-90 and DbM187-195 tetramer-binding CD8+ T cells in the lungs of mice infected as adults or neonates 7 days earlier with replication-defective rAd5 vector expressing M and M2 in the form of a fusion protein. (B) Epitope-specific CD8+ T cell response ratios. Data are representative of two independent experiments with 5 mice/group. Error bars represent the SEM.
Figure 5.
TCR Vβ screening of naïve and epitope-specific CD8+ T cells in adult and neonatal mice.
(A) TCR Vβ antibody screen of CD8+ T cells from the spleen of naïve adult and naïve neonatal CB6F1 mice. Samples were stained with surface phenotype antibodies, and a panel of FITC-labeled Vβ-specific antibodies. (B) TCR Vβ antibody screen of epitope-specific (KdM282-90 and DbM187-195) CD8+ T cell populations in the lungs of RSV-infected adults and neonates. Lung lymphocytes were stained with surface phenotype antibodies, epitope-specific tetramer, and Vβ-specific antibodies. All TRBV regions are listed using IMGT nomenclature. Data represents two independent experiments performed with pooled naïve splenic or lung lymphocytes.
Figure 6.
Single cell TCRβ sequences from RSV-specific CD8+ T cell populations.
(A) TCR Vβ usage in single CD8+ T cells sorted with KdM282-90 (left panels) or DbM187-195 (right panels) tetramers from RSV-infected adult (upper panels) and neonatal (lower panels) CB6F1 mice (IMGT nomenclature). (B) CDR3β consensus sequences generated on http://weblogo.berkeley.edu/for single CD8+ T cells sorted with KdM282-90 (left panels) or DbM187-195 (right panels) tetramers from RSV-infected adult (upper panels) and neonatal (lower panels) CB6F1 mice. Single tetramer-positive cells were sorted and sequenced from two mice/group/epitope.
Figure 7.
Epitope-specific CD8+ T cell responses in wild-type and TdT-/- adult CB6F1 mice.
(A) KdM282-90 and DbM187-195 tetramer-positive CD3+CD8+ in the lungs at day 7 post-infection of wild-type (WT) and TdT deficient (TdT-/-) CB6F1 mice. (B) Epitope-specific CD8+ T cell response ratios in infected WT and TdT-/- mice. (C) TCR Vβ antibody screening of pooled tetramer-positive CD8+ T cells from the spleen of infected WT and TdT-/- mice (IMGT nomenclature). Data in panels (A) and (B) are representative of two independent experiments with 5-6 mice/group. Error bars represent the SEM.
Figure 8.
Epitope-specific CD8+ T cell precursor frequency analysis in adult and neonatal mice.
(A) Precursor frequencies of epitope-specific CD8+ T cells in naïve adult and neonatal mice as determined by the double tetramer enrichment method. (B) KdM282-90/DbM187-195 precursor frequency ratios in naïve adults and neonates. (C) Epitope-specific memory CD8+ T cell precursor frequencies in adult mice infected one month previously with RSV. (D) Memory CD8+ T cell precursor ratios. Data are representative of several independent experiments with 5-12 mice/group. Error bars represent the SEM.
Figure 9.
Age at infection determines CD8+ T cell epitope hierarchy.
(A) KdM282-90 and DbM187-195 epitope-specific CD8+ T cell responses determined by tetramer staining at day 7 post-infection of mice infected with RSV at the indicated day of life. (B) Epitope-specific CD8+ T cell response ratios at day 7 post-infection of mice infected with RSV at the indicated day of life. (C) Viral titers in the lung at day 4 post-infection of mice infected with RSV at the indicated day of life. Data represent two independent experiments with 4-8 mice/group. Error bars are the SEM.
Figure 10.
Intrinsic CD8+ T cell factors contribute to epitope hierarchy post-infection.
(A) KdM282-90 and DbM187-195-specific CD8+ T cells in the lungs of control neonatal mice, and neonatal mice that received congenic transfer of naïve adult CD8+ T cells. (B) Epitope-specific CD8+ T cell response ratios in control neonatal mice and neonatal mice that received congenic transfer of naïve adult CD8+ T cells. Data are representative of several independent experiments with 3-10 mice/group. Error bars represent the SEM.