Fig 1: OVA-releasing scaffolds implanted in vaccinated mice enhance TH2 responses after ischemic injury.(A) Experimental setup with (left) BALB/c mice undergoing (right) timeline of procedures involving vaccination and boost with OVA/ALUM, followed by hindlimb ischemia induction and scaffold implantation. (B to J) In these figures, naïve + OVA denotes mice receiving no vaccination and implanted with an OVA-containing scaffold, vacc + blank denotes mice receiving vaccination with OVA/ALUM and a blank scaffold, and vacc + OVA denotes mice receiving vaccination with OVA/ALUM and an OVA-containing scaffold. (B) Number of CD4+ T cells (TCR-β+/CD4+ cells) per milligram of tissue in the ischemic upper leg at days 4 and 7 after ischemic ligation in the various treated groups. (C) Representative images of IL-5 enzyme-linked immunospot (ELISPOT) assay, where cells derived from the ischemic upper leg were cultured in the presence of splenocytes presenting either OVA or CT26-gp70 peptide as an irrelevant peptide control. (D) Quantification of OVA-specific IL-5 spot-forming cells per milligram of tissue determined by subtracting the number of IL-5 spot-forming cells in the CT26-gp70 condition from that in the OVA condition. (E to G) Concentrations of (E) IL-5, (F) IL-10, and (G) IFN-γ produced from OVA-stimulated cells isolated from the ischemic upper leg 7 days after ischemic ligation (n = 6 mice for naïve + OVA; n = 4 mice for vacc + blank; and n = 4 mice for vacc + OVA). (H to J) Number of (H) macrophages (F4/80+ cells), (I) M2a macrophages (F4/80+/CD206+ cells), and (J) eosinophils (Siglec-F+/CD11b+ cells) per milligram of tissue in the ischemic upper leg at days 4 and 7 after ischemic ligation in the various treated groups. For day 4 data in (B) to (D) and (H) to (J), n = 5 mice for naïve + OVA, n = 6 mice for vacc + blank, and n = 6 mice for vacc + OVA. For day 7 data in (B) to (D) and (H) to (J), n = 3 mice for naïve + OVA, n = 6 mice for vacc + blank, and n = 5 mice for vacc + OVA. Data are presented as means ± SD. Significance is denoted by *P ≤ 0.05, **P ≤ 0.01, or ***P ≤ 0.001 by one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test.
Fig 2: Early-life viral infection results in secondary response with increased CD4+ Th2 signature.(A) Representative flow plots and quantification of intracellular GATA3 staining in CD4+ T cells isolated from the lung at day 7 after rechallenge showing increased levels in nV/aV mice. (B–D) Intracellular staining of IL-4, IL-5, and IL-13 production after ex vivo HMPV peptide stimulation in lung CD4+ T cells 7 days after rechallenge. *P < 0.05, **P < 0.01, ***P < 0.005 by 2-tailed Student’s t test.
Fig 3: Early JAK2 inhibition reduces asthma pathology.(A) UMAP visualization of Jak2 expression, showing upregulation in green circle corresponding to the Th2 cluster. (B) Mice were treated with fedratinib (120 mg/kg) once prior to rechallenge followed by BID dosing for the first 4 days after rechallenge. DMSO was used for vehicle control treatment. Created with BioRender.com. (C) Weight changes after rechallenge with HMPV. (D) Quantification of eosinophils 7 days after rechallenge. *P < 0.05 by Mann-Whitney U test. (E) N217-specific IL-4 and IFN-γ production after HMPV rechallenge, with a trend (P = 0.09, 2-tailed Student’s t test) toward reduced IL-4 production in JAK2i-treated mice. (F and G) IL-4, IL-5, IL-13, IL-2, IFN-γ, and GM-CSF protein quantity from whole lung homogenate. (H) Representative images of PAS staining demonstrating mucus production in large airways with reduction in JAK2i. Scale bar: 250 μm. (I) Quantification of PAS+ staining as percentage of total cell detections (left) and lung area (right). *P < 0.05 by 2-tailed Student’s t test.
Fig 4: Early-life viral infection leads to skewed secondary response recapitulating asthma pathology.(A) Schematic of primary neonatal HMPV with secondary rechallenge in adulthood (nV/aV) or primary adult HMPV with rechallenge (aV/aV). Mice were euthanized at day 7 after rechallenge. Created with BioRender.com. (B) Weight loss was observed in both groups after rechallenge (n = 4–7/group). (C) IL-4, IL-5, IL-13, and CCL11 protein quantity from whole lung homogenate. **P < 0.01, ****P < 0.0005 by 2-tailed Student’s t test. (D) Representative flow plot of lung eosinophil identification (left) and enumeration (right) from lung homogenate and bronchoalveolar lavage (BAL) 7 days after rechallenge. *P < 0.05, **P < 0.01 by 2-tailed Student’s t test. (E) Periodic acid–Schiff (PAS) staining on FFPE lung tissue at day 7 after rechallenge showing mucus plugging in nV/aV mice. Scale bar: 100 μm (F) Quantification of PAS+ staining as percentage of total cell detections (left) and lung area (right). *P < 0.05, **P < 0.01 by 2-tailed Student’s t test. (G) FlexiVent analysis demonstrating airway hyperresponsiveness in nHMVP/aHMPV mice at 50 mg/mL methacholine administration. n = 2, 6, 5 for mock/mock, nV/aV, and aV/aV groups, respectively. ****P < 0.0005 by 2-way ANOVA with Dunnett multiple-comparison test.
Fig 5: Single-cell RNA-seq of TRM populations after neonatal or adult HMPV infection.(A) Uniform manifold approximation and projection (UMAP) visualization of lung TRM populations isolated by flow-sorting 5 weeks after neonatal or adult HMPV infection demonstrating 9 clusters. (B) Expression of Th1, Th2, Treg, and Th17 transcription factors (e.g., Tbx21, Gata3, Foxp3, Rorc) and cytokines (Ifng, Il5, Il10, Il17a) demonstrating populations consistent with these established subsets. (C) Expression of markers corresponding to other cell states, including Pdcd1 (PD-1+), Sell (CD62L, naive), Trgv2 (innate-like T cell), and cycling (Mki67). Expression patterns in B and C and Supplemental Figure 2 were used to manually annotate the 9 clusters shown in A. (D) UMAP visualization of TRM clusters split by group (NeoTRM vs. AdultTRM). (E) Volcano plot of differentially expressed genes in pseudo-bulked NeoTRM versus AdultTRM cells. Genes displayed have Padj < 0.01 and log2FC (≤0.25 or >0.25). (F) Proportion testing of individual clusters in NeoTRM versus AdultTRM. Significance defined as FDR < 0.05 and abs(log2FC > 0.58). (G) Clonotype analysis of TCR sequencing with each bar representing count of an individual TCR. Naive/Tcm cells had no more than 3 of the same TCR per cluster (dashed line). (H) Clonotype expansion in Th1, PD-1+ Th1, Th2, and Th17 clusters.
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