Fig 1: Regulation of the Effector T Cell Response by Antigen Receptor Signaling(A) Ly5.1 Nur77-GFP OT-I CD8 T cells were adoptively transferred into congenic Ly5.2 C57BL/6 mice and vaccinated IN next day with OVA protein formulated in the indicated adjuvants. At days 2, 5, or 8 PV, cells from lymph nodes and lungs were stained with Kb/SIINFEKL tetramers, anti-Ly5.1, anti-Ly5.2, anti-CD8, and anti-CD44 antibodies. The GFP MFIs in donor Ly5.1+ve OT-I CD8 T cells were quantified by flow cytometry.(B) Wild-type non-transgenic (WT) and transgenic KLF2-GFP mice were vaccinated with NP protein formulated in adjuvants, as in (A). At day 8 PV, lung cells were stained with anti-CD8, anti-CD44, and Db/NP366 tetramers. The overlay histogram shows GFP fluorescence (MFI) for the gated tetramer-binding CD8 T cells from WT (black) and KLF2-GFP transgenic (red) mice.(C) B6 mice were vaccinated with NP protein formulated in various adjuvants, as in (A). At day 8, PV lung cells were stained with anti-CD8, anti-CD44, anti-PD-1, and Db/NP366 tetramers. Plots show the percentages of PD-1+ve cells among the gated Db/NP366 tetramer-binding CD8 T cells.(D) Statistical correlation analysis between the percentages of PD-1+ve CD8 T cells and the percentages of tetramer+ve CD8 T cells at day 8 PV.Data are pooled from two independent experiments or represent one of two independent experiments. Comparisons were made using one-way ANOVA test with Tukey-corrected multiple comparisons. For (D), we used two-way ANOVA, Student’s t test, and simple regression analysis; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Fig 2: PTBP1 is essential for the expansion and differentiation of CD8 T cells in vivo. (A) Proportions of CD45.2+ cells (among live eFluor780– lymphocytes) identified by flow cytometry in the blood of B6.SJL (CD45.1+) mice which received 1000 naïve CD8 T cells from OT1 control (CD45.2+ OT1Tg ) or OT1 P1KO (CD45.2+ OT1Tg Cd4CreTg Ptbp1fl/fl ) mice and were infected with attLm‐OVA 1 day after OT1 cell transfer. Each symbol shows the mean ± SD of four mice per group. Data shown are from one experiment. (B) Note that 75,000 naïve CD8 T cells from CD45.1+ OT1Tg Cd4CreTg and 75,000 naïve CD8 T cells from CD45.2+ OT1Tg Cd4CreTg Ptbp1fl/fl P1KO mice were adoptively co‐transferred into CD45.1+ and CD45.2+ double‐positive mice, which were infected with attLm‐OVA 1 day after cell transfer. Shown are the proportions of CD45.1+ or CD45.2+ cells among live (eFluor780–) CD8a+ splenocytes at different days postinfection. (C) Numbers of CD45.1+ or CD45.2+ cells among live (eFluor780–) CD8a+ splenocytes per mouse at different days postinfection treated as shown in (B). p adj values shown are from Sidak's multiple correction test after two‐way ANOVA from the log‐transformed data. (D) Cell Trace Violet profiles of CD45.1+ or CD45.2+ CD8 OT1 cells (identified as live eFluor780– and CD8a+) in spleens of CD45.1 and CD45.2 double‐positive mice, which received an adoptive co‐transfer of 200,000 naïve CD45.1 control OT1 cells (from OT1Tg CD4CreTg mice) and 200,000 naïve CD45.2+ P1KO OT1 cells (from OT1Tg CD4CreTg Ptbp1fl/fl mice) 3 days postinfection with attLm‐OVA. Graph shows gMFI of CD45.1+ or CD45.2+ CD8 OT1 cells shown on the left. Each point shows data from one mouse. Lines show arithmetic means. p‐value from a two‐tailed paired Student's t‐test is shown. Data are from one experiment with five CD45.1 and CD45.2 double‐positive recipient mice. (E) Percentages of active caspase 3+ cells (identified as shown in Supporting Information S1D) on different days after att‐LmOva infection in mice that received an adoptive co‐transfer of control and PTBP1‐deficient OT1 naïve CD8 T cells as described in (B). p values shown are from two‐tailed paired Student's t‐tests done on the log transformed data. Lines show arithmetic means. Data shown are from one experiment with four mice analyzed per day. Each point shows data from an individual mouse. (F) Cytokine and granzyme B staining of CD45.1+ PTBP1+ (black histograms) or CD45.2+ PTBP1– (blue histograms) live (eFluor780–) CD8+ splenocytes of mice treated as shown in (B) and re‐stimulated with 50 nM SIINFEKL peptide for 3 h in the presence of brefeldin A. Orange histograms show PTBP1+ CD8a+ splenocytes from an OT1Tg mouse. Data shown are from a representative mouse per day. Gates show positive populations. (G) Proportions of cytokine expressing cells identified as shown in (F) and geometric mean fluorescence intensity (gMFI) of the staining within cells identified as positive. Lines show arithmetic means. Data shown are from one experiment with four mice analyzed per day. p adj values shown are from Sidak's multiple correction test after two‐way ANOVA. (H) gMFI of granzyme B staining among CD45.1+ PTBP1+ or CD45.2+ PTBP1– live (eFluor780–) CD8+ splenocytes. Lines show arithmetic means. Data shown are from one experiment with four mice analyzed per day. p adj values shown are from Sidak's multiple correction test post two‐way ANOVA. (I) Proportions of KLRG1+ cells among CD45.1+ PTBP1+ or CD45.2+ PTBP1– cells in spleens of mice treated as described in (B). Gating strategy is shown in Supporting Information Fig. S1E. Lines show arithmetic means. Data shown are from one experiment with four mice analyzed per day. p adj values shown are from Sidak's multiple correction test after two‐way ANOVA.
Fig 3: In the blood, Bdpy-ML incorporates differently into cell populations and is largely found in plasma compartment. (A) Incorporation of Bdpy-ML as measured by FACS into erythrocytes (Erythro, CD235a+ cells) and leukocytes (Leuko, CD45+ cells) after 16 h of incubation in total human blood. Data are mean fluorescence intensities (MFI) ±SD normalized to cell size (FCS corrected) of triplicates. (B) Incorporation of Bdpy-ML into CD45+ blood cells, including T cells (T), NK cells (NK), B cells (B), and monocytes (Mono), after 16 h of incubation in total human blood. Data are mean fluorescence intensities (MFI) ±SD normalized to cell size of replicates from 3 donors and are representatives of two independent experiments. Test one-way Anova, *p < 0.1, ***p < 0.001, ****p < 0.0001 (C) Recovery of Bdpy-ML in plasma fraction as measured by spectrofluorimetry, 16 h after addition of either 50 ng or 500 ng of Bdpy-ML or of 500 ng of Bdpy-ML in presence of 500 ng of natural mycolactone (ML) into total human blood. Data are expressed as percentage of recovery compared to plasma spiked with the same concentrations of Bdpy-ML and are means ± SD of 6 donors.
Fig 4: Regulation of Vaccine-Induced CD8 T Cell Memory and Protective Immunity by CD4 T CellsGroups of C57BL/6 mice were vaccinated with NP+ADJ+GLA, as in Figure 1, and treated with isotype control antibodies (non-depleted) or anti-CD4 antibodies (CD4-depleted) i.v. and IN on days −1, 0, and 1 relative to prime and boost vaccination. T cell memory in lungs (A–F) and protective immunity to influenza A virus (G–P) was determined at 80 days PV.(A–F) T cell memory in lungs at day 80 PV. To stain for vascular cells, mice were injected i.v. with anti-CD45.2 antibodies, 3 min prior to euthanasia. Lung cells were stained directly ex vivo with Db/NP366 or I-Ab/NP311 tetramers along with the indicated antibodies. For cytokine analysis, lung cells were stimulated with NP366 or NP311 peptide for 5 h before intracellular staining.(A) FACS plots are gated on CD4 T cells and show NP311-specific, tetramer-binding memory CD4 T cells only in non-depleted mice.(B) NP366-specific tetramer-binding memory CD8 T cells in lungs of non-depleted and CD4 T-cell-depleted mice.(C) Expression of tissue residency markers on NP366-specific, tetramer-binding memory CD8 T cells in lungs.(D) Percentage of vascular (CD45.2+ve) and non-vascular (CD45.2−ve) cells among NP366-specific, tetramer-binding memory CD8 T cells in lungs.(E) Percentages of IFN-γ- and IL-17-producing, NP366-specific cells among CD8 T cells in lungs.(F) Calculated proportions of IFN-γ- and/or IL-17-producing cells among cytokine-producing, peptide-stimulated, IFN-γ+IL-17 NP366-specific CD8 T cells.(G–P) At day 80 after booster vaccination, non-depleted and CD4 T-cell-depleted mice were challenged IN with PR8/H1N1 influenza A virus; recall virus-specific CD8/CD4 T cell responses and viral load in lungs were assessed at day 6 after challenge.(G) Percentages of NP366-specific, tetramer-binding cells among CD8 T cells in lungs.(H) Percentages of NP366-specific, tetramer-binding CD8 T cells in vascular and non-vascular lung compartment.(I) Percentages of NP311-specific, tetramer-binding cells among CD4 T cells in lungs.(J) Expression of tissue residency markers on NP366-specific, tetramer-binding CD8 T cells.(K) Chemokine receptor and transcription factor expression in NP366-specific CD8 T cells in lungs.(L) Granzyme B expression by NP366-specific CD8 T cells directly ex vivo.(M) Percentages of IFN-γ- and IL-17-producing, NP366-specific CD8 T cells.(N) Relative proportions of IFN-γ- and/or IL-17-producing cells among total IFN-γ plus IL-17-producing, peptide-stimulated, NP366-specific CD8 T cells.(O) Viral titers in lungs at day 6 after challenge.(P) Body weight measured as a percentage of starting body weight prior to challenge.Data are pooled from two independent experiments. Comparisons were made using one-way ANOVA test with Tukey-corrected multiple comparisons; ∗p < 0.1, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Fig 5: Endothelin-1 Expression Highly Correlates with Localization of IAHCs(A) Expression overlap between EDN1, CDH5, and REN1. The arrowhead indicates a REN1+ cell below the CDH5+ endothelium. The images in (Ai) show a magnification of the boxed region in (A).(B) Expression of REN+ cells enveloping the endothelium of an Ao V branching vessel (BV) directed toward the Mn. The images in (Bi) show a magnification of the boxed region in (B).(C and D) Immunostaining highlighting V CDH5+Runx1+CD45+ IAHCs (C’ and D’) and, on the sister section, a higher EDN1 signal in a corresponding position (C’’ and D’’). Arrowheads indicate positions of IAHCs.(E) Representative binary image of EDN1 expression across the Ao with EDN1 hotspots (pixels > 300, 2,048 × 2,048 pixel image) numbered and outlined in red. A line divides the AoD (top) from the AoV (bottom). The box-and-whisker plot shows the percentage of EDN1 hotspots found in the AoV or AoD in each section (n = 14). p < 0.01, t test.(F) CL of rounded EDN1-expressing cells attached to the CDH5+ endothelial lining (arrows). Images in (Fi) show a magnification of the boxed region in (F).(G) Correlation between the position of Runx1+ IAHCs in each section with the position of EDN1 hotspots (R, correlation coefficient).For (A)–(C), (E), and (F), protein or RNA expression is indicated by non-italicized and italicized names, respectively. SMA, superior mesenteric artery. The D-V axis is indicated. Images show transverse sections of CS15–CS16 embryos. Scale bars, 50 µm.
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