Supplementary Figures
Supplementary Figure 1. Gating strategy for thoracic aorta single cell suspension at 30 and 60 (d.p.i). T cells were gated from live cells, which were gated from CD45+ Lymphocytes. CD4+ and CD8+ T cells were further gated into CD44+, CD69+, naïve cells, TCM cells, and TEM cells. CD8+ T cells were also gated to reveal CD44+IAV-Specific CD8+ T cells, and the TCM or TEM cell phenotypes within the IAV-Specific CD8+ T cell population.
Supplementary Figure 2. IAV infection in pregnancy is associated with increased pup deaths. Pregnant mice infected with PBS or HKx-31(x-31) at 104 PFUs was assessed for the following pregnancy outcomes: gestation at delivery, litter sizes and number of pup deaths 24 h after birth. Data shown represent PBS, n = 44 litters and X-31, n = 56 litters across the study. Results are presented as mean ± SEM. Unpaired Student’s t-test was used for statistical analysis against respective PBS controls. ****P < 0.0001.
Supplementary Figure 3. Area under the curve responses of aorta in IAV infected mice. Vascular function was measured in isolated thoracic aortic rings of non-pregnant mice infected with PBS or HKx-31(x-31) at 104 PFUs for assessment of (A) endothelium dependent vasodilation to acetylcholine (Ach) and (B) endothelium independent vasodilation to sodium nitroprusside (SNP; B) at 12, 30 and 60 d.p.i. Area under the curve was calculated to determine the total vascular response to Ach and SNP. Data shown represent PBS, n = 6; X-31, n = 5; of at least two independent experiments. Results are presented as mean ± SEM. Unpaired Student’s t-test was used for statistical analysis against their respective PBS controls. *P < 0.05; **P < 0.005.
Supplementary Figure 4. CD4+ and CD8+ T cells are not persistently elevated in the aorta of non-pregnant mice. Non-pregnant and pregnant 8-12 week old mice were inoculated with PBS or HKx-31 (X-31) at 104 PFUs. Mice were culled at 30 and 60 d.p.i and aorta single cell suspensions were assessed via flow cytometry for the proportion of live CD45+ leukocytes that are CD4+ or CD8+ T cells, the number of CD4+ and CD8+ T cells, the proportion of CD8+ T cells that are CD44+ and DbPA224 tetramer+ and number of CD8+ CD44+ DbPA224 tetramer+ T cells recovered from aortic tissue. All data shown represent pregnant PBS, n = 4-7; pregnant X-31, n = 4-10; of at least two independent experiments. All cell populations are measured as absolute number of cells per 25,000 counting beads. Results are presented as mean ± SEM. Unpaired Student’s t-test was used for statistical analysis against respective PBS controls. *P < 0.05.
Supplementary Figure 5. Naïve, TCM, TEM and putative TRM subsets within CD4+ and CD8+ T cell populations in the aorta post viral clearance. Non-pregnant and pregnant 8-12 week old mice were infected with PBS or HKx-31 (X-31) at 104 PFUs and culled at 30 and 60 d.p.i. Aorta single cell suspensions were assessed via flow cytometry for the numbers of CD4+ naïve (CD44-CD62L+), TCM (CD44+CD62L+), TEM CD44+CD62L-) cells and putative CD4+ TRM (CD44+CD69+) or numbers of CD8+ naïve (CD44-CD62L+), TCM (CD44+CD62L+), TEM (CD44+CD62L-) and putative CD8+ TRM (CD44+CD69+) cells in the aorta. Data shown represent pregnant PBS, n = 4-7; pregnant X-31, n = 4-10; of at least two independent experiments. All cell populations are measured as absolute number of cells per 25,000 counting beads. Results are presented as mean ± SEM. Unpaired Student’s unpaired t-test was used for statistical analysis against respective PBS controls. *P < 0.05.
