Fig 2: Target engagement and specificity of BP79.(A) Proximity ligation assay (PLA): TSLP binds to TSLPR and forms a ternary complex with IL-7Rα at the cell surface initiating intracellular signaling. PLA was performed to verify the inhibition of TSLP-mediated ternary complex formation via BP79. Primary keratinocyte cells were treated with the TSLP inhibitors and stimulated with TSLP. Cells were fixed and the interaction between TSLPR and IL-7Rα was observed with PLA assay. BP79 inhibited the TSLP:TSLPR:IL-7Rα ternary complex formation. Images were counted using ImageJ (n = 20). Scale bar = 50 μm. (B) In vitro thermal shift assay. BP79 thermally stabilized TSLPR up to 45 °C. (C) To determine if BP79 decreases the cytokine expression through direct binding of upstream kinases such as JAK1/2 or other kinase targets, BP79 was screened against 97 kinase targets using the KINOMEscan™ platform (radioligand displacement assay performed by Eurofins Discovery Services). Data information: All data are presented as mean ± SEM. n represents the number of biological replicates. Statistical analysis was performed using Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Source data are available online for this figure.

Fig 3: Dose-dependent screen of BP79 analogs in CD4 + T cells.(A) IL-13 expression in primary CD4 + T cells after treatment with 111 nM, 333 nM, and 1000 nM of inhibitor for 36 h. (B) IL-4 expression primary CD4 + T cells after treatment with 111 nM, 333 nM, and 1000 nM of inhibitor for 36 h. Bars represent mean percent cytokine expression relative to the DMSO control, n = 3. Non-treated (NT), CD3/CD28 stimulated (CD3/CD28), CD3/CD28 and TSLP-stimulated (CD3/CD28 + TSLP), and vehicle (CD3/CD28 + TSLP + DMSO) controls were included. (C) Dose–response curves of BP79, which were used to calculate the IC50 value and HillSlope (n = 3). Data information: All data are presented as mean ± SEM. n represents the number of biological replicates. Statistical analysis was performed using Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig 4: Full panel images of proximity ligation assay (PLA).PLA was performed to check the inhibition of TSLP-mediated ternary complex formation with BP79. Primary keratinocyte cells were treated with the TSLP inhibitors and stimulated with TSLP. Cells were fixed and the interaction between TSLPR and IL-7Rα was observed with PLA assay. C7 and BP79 inhibited the TSLP:TSLPR:IL-7Rα ternary complex formation. Scale bar = 50 μm.

Fig 5: Development & utilization of an atopic diseases-organ chip for preclinical BP79 testing.(A, B) Experimental setup of the co-cultivation of normal or atopic-like skin models with 3D normal bronchial epithelial models in a microfluidic two-organ chip. BP79 and tacrolimus were topically applied onto the skin (disease) model. (C) H&E staining of co-cultivated skin (normal and diseased) and lung tissue models w/ or w/o topical application of BP79 or tacrolimus. (D) Topical application of BP79 significantly reduced IL-13, IL-4, TSLP and periostin expression as detected via ELISA. Scale bar = 50 μm. (E) Relative mRNA expression of TSLP, filaggrin, keratin 5, keratin 14, and keratin 10 in atopic-like skin and bronchial lung tissue models following topical treatment with BP79 and tacrolimus determined via quantitative RT-PCR (n = 3). GAPDH served as a housekeeping gene.