Fig 2: Intragastric B. vesicularis aggravates MC903-induced AD-like dermatitis in mice(A) Experimental design. Mice were intragastrically administered with antibiotic cocktail daily for 3 days, then gavaged with 200 μL of B. vesicularis (1 × 109 CFU) daily for 7 days. Intragastric PBS was used as control. Thereafter, gavage was continued, and mice were topically treated with 1 nmol of MC903 and 20 μg/μL of OVA daily for 15 days to produce the AD mouse model.(B) Gross appearance of the ears on day 27.(C) Representative H&E staining for the skin lesion (scale bars, 250 μm). Enlarged pictures show the infiltration of eosinophils (scale bars, 50 μm).(D) Dynamic changes of ear thickness at the indicated time points. n = 6.(E) Ear thickness of the epidermis (EM) on day 27. n = 6.(F) Numbers of infiltrating eosinophils counted from H&E staining. n = 6.(G) Levels of serum total IgE at baseline and day 27 as measured by ELISA. n = 5.(H) CD11c+MHCII+CD103+ DCregs from the CLNs and MLNs, as measured by flow cytometry. Representative dotplots and aggregated statistics of biological replicates are shown. n = 5 or 6.(I) Levels of TGF-β and IL-6 in the supernatants of CD11c+ DC culture, as measured by ELISA. n = 5.(J and K) CD4+RORγt+ Th17 cells from the CLNs, MLNs, and spleens, as measured by flow cytometry. Representative dotplots show the frequency of gated RORγt+ Th17 cells among CD4+ T cells (J). Aggregated statistics of biological replicates are shown in (K). n = 5 or 6.(L) Th17/Treg ratios from the CLNs, MLNs, and spleens, as measured by flow cytometry. n = 5 or 6.Data are shown as mean ± SEM (D–I, K, and L). Unpaired two-tailed t test: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. See also Figures S5 and S6.

Fig 3: Pharmacological and genetic inhibition of TGF-β signaling interferes with in vitro TRM generation(A) CD8 T cells co-cultured with VEOs were treated with TGF-β signaling inhibitor, SB431542 (10 μM), or vehicle control for 7 days. Representative flow plots of live CD8β+ T cells are shown.(B and C) Percentages of TRM phenotype cell are enumerated. CD8 T cells maintained alone are included as a control.(D) CD8 T cells co-cultured with VEOs were treated with increasing concentrations of an inhibitor of TGF-β activating αV integrins, CWHM-12 (10 nM–10 μM), or vehicle for 12 days. Representative flow plots of live CD8β + T cells are shown.(E) Percentages of CD69+CD103+ phenotype cells are enumerated.(F) TGF-β-receptor-deficient CD8 T cells fail to adopt TRM phenotype in the VEO co-culture model. Wild-type (WT; CD45.1+CD45.2+) and TGF-βRII-deficient P14 (KO, CD45.1+CD45.2−) CD8 T cells were activated and embedded in BME containing VEOs at a 1:1 ratio. Representative flow plots 12 days after the co-incubation are shown. Total P14 CD8 T cells and the ratio of WT and KO CD8 T cell percentages retained as well as their associated phenotypes after 12 days are shown.(G and H) Comparison of CD69+CD103+ CD8 T cells and H. p2rx7+ CD8 T cells between WT and KO groups. Data are representative of two repeats with n = 4–6/condition. Bars indicate mean ± SEM.One-way ANOVA with Tukey’s multiple comparison test (B, C, and E). Wilcoxon matched-pairs signed rank test (G and H). *p < 0.05 and ****p < 0.0001.

Fig 4: B. vesicularis attenuates AIT efficacy in AD mice(A) Experimental design. Mice were intragastrically administered with antibiotic cocktail daily for 3 days and then gavaged with 200 μL of B. vesicularis (1 × 109 CFU) daily for 7 days. PBS was used as control. Thereafter, gavage was continued, and mice were topically treated with 1 nmol of MC903 and 20 μg/μL of OVA daily for 14 days to establish the AD mouse model. Once the dermatitis was fully induced, the same dose of MC903 and OVA was applied every other day for 5 days to maintain the skin inflammation. Starting on day 10 of dermatitis induction, mice were injected subcutaneously with 200 μL of OVA solution once every 4 days for three consecutive times, using normal saline as control.(B) Gross appearance of the ears and representative H&E staining with enlarged picture showing eosinophils on day 30. Scale bars, 50 μm.(C) Dynamic changes of ear thickness at the indicated time points. n = 6.(D) Ear thickness of the epidermis (EM) on day 30. n = 6.(E) Numbers of infiltrating eosinophils counted from H&E staining. n = 5 or 6.(F) Levels of serum total IgE and OVA-specific IgE at baseline and day 30 as measured by ELISA. n = 5 or 6.(G) CD11c+MHCII+CD103+ DCregs from the CLNs and MLNs, as measured by flow cytometry. Representative dotplots and aggregated statistics of biological replicates are shown. n = 5 or 6.(H) Mean fluorescence intensity (MFI) of PD-L1, CD86, or MHCII on CD11c+MHCII+ DCs from the CLNs and MLNs, as measured by flow cytometry. n = 5 or 6.(I) Th17/Treg ratios from the CLNs, MLNs, and spleens, as measured by flow cytometry. n = 5 or 6.Data are shown as mean ± SEM (C–I). Unpaired two-tailed t test: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. See also Figure S7.

Fig 5: VEOs support viral replication, and the co-culture system is amenable to genetic perturbation(A) 7-day-old VEOs were mock infected or infected with HSV-1 K26GFP or HSV-2(333ZAG)-GFP. Wells containing infected and uninfected cells were visualized 24 h post-infection using confocal microscopy. Representative images are shown. Scale bar: 50 μm.(B) In-vitro-activated P14 CD8 T cells were retrovirally transduced with Runx3-EGFP-expressing vector or control-mCherry-expressing vector. Equivalent numbers of cells were cultured with VEOs for 10 days, and their ability to form TRMs was tested by flow cytometry. Representative flow plots of total transgene-positive P14 cells are shown on the left, and the levels of CD69 and CD103 on the two reporter-positive populations are shown on the right.(C) Bar graph comparing percentage of CD69+CD103+ cells among the two transduced populations. Data are representative of two repeats with at least n =3 wells/condition. Bars indicate mean ± SEM. Student’s t test. ****p < 0.0001.