Single-cell atlas of patient-derived cervical organoids uncovers epithelial immune heterogeneity and intercellular crosstalk during Chlamydia infection

The uterine cervix is a critical mucosal interface that balances immune defense and reproductive function, yet how its distinct epithelial compartments coordinate responses to infection remains unclear. Here, we integrate patient-derived 3D cervical organoids, single-cell transcriptomics and native tissue analysis to construct a high-resolution atlas of epithelial cell diversity and immune dynamics during Chlamydia trachomatis infection. We demonstrate that cervical organoids precisely mirror native tissue at both transcriptional and cellular levels, identifying epithelial subtypes with region-specific immune specializations. Upon infection, ectocervical epithelia reinforce barrier integrity, whereas endocervical epithelia, particularly uninfected bystander cells, exhibit extensive transcriptional reprogramming characterized by robust interferon activation, antigen presentation, and antimicrobial defense. Infection profoundly reshapes epithelial intercellular communication, positioning bystander cells as central signaling hubs that coordinate immune responses and tissue regeneration. Our findings highlight a sophisticated epithelial-intrinsic immune network critical for cervical mucosal defense and establish a physiologically relevant platform for studying human host-pathogen interactions and guiding targeted mucosal therapies against reproductive tract infections and pathologies. Overall design: Human cervical organoids were cultured as described in Chumduri et al. 2021, Nature Cell Biology. Ecto-and endocervical organoids were infected with Ctr-L2-GFP strain, and then organoids were harvested, and Matrigel was removed. For the human endocervix, organoids were broken down before infection by passing the organoid suspension through a 26G needle 4 times. Organoids were infected at MOI of 5 in an infection medium for 2 hr at 37°C on a shaker (~100 rpm). After infection, organoids were washed, seeded on Matrigel, and allowed to grow in a 3D media for 36 hr for single-cell RNA-seq analysis. Replicates (3 biological replicates of human ecto- and 2 biological replicates from endocervix) were pooled, and cell multiplexed using Cell Multiplexing Oligos (CMOs) based on their tissue region using 10x Genomics 3' CellPlex and Single-Cell 3' v3.1 RNA-seq kits. CMO-labeled cells were pooled at an equal ratio, made the cell concentration to 2000 cell/µL, and proceeded for the library preparation. Single cells were partitioned into nanolitre-scale Gel-Bead-In-EMulsions (GEMs) using a 10x Chromium Controller. The library was prepared using Single-Cell 3' reagent kit v3 for reverse transcription, cDNA amplification, and library construction according to the manufacturer's protocol. Sequencing was performed in paired-end mode with an S1 100-cycles kit using Novaseq 6000 sequencer (Illumina)