Abstract Rationale Acute cellular rejection (ACR) is a leading risk for chronic lung allograft dysfunction (CLAD) following lung transplantation. Early recognition of ACR is essential to prevent irreversible graft injury. However, current diagnostic strategies rely on histopathologic interpretation of transbronchial biopsies, which are invasive and limited by sampling variability and modest sensitivity. Molecular profiling of the lower airway may provide a more direct readout of alloimmune activity. We therefore performed transcriptomic profiling of bronchoalveolar lavage (BAL) samples to identify host gene expression signatures that distinguish ACR and potentially precede histologically appreciable rejection. Methods We analyzed 79 BAL samples collected during surveillance bronchoscopy from 53 lung transplant recipients: 28 with biopsy-confirmed ACR and 25 age-, sex-, and UNOS listing diagnosis group-matched non-ACR controls. In a subset of patients (n=26), repeated sampling was performed at one month (“baseline”) and at time of ACR diagnosis, or at similar timepoints in non-ACR subjects, to facilitate longitudinal comparisons. RNA sequencing was performed on BAL cell pellets, and host reads were aligned to the human reference genome using Bowtie2. Differential gene expression analysis was conducted using edgeR (R) with false discovery rate correction. Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). Results At baseline, recipients who later developed biopsy-confirmed ACR exhibited enrichment of pathways involved in cilium movement, axoneme assembly, and microtubule-based transport compared with those who never experienced rejection. In the sequential analysis, which compared longitudinal samples from patients with active ACR to their matched controls, these same pathways were significantly upregulated, with higher normalized enrichment scores (NES), indicating persistent or augmented activation of ciliary and cytoskeletal gene programs during ongoing rejection (Figure 1A). Consistent with these findings, IPA revealed activation of RHO GTPase signaling and pathogen-induced cytokine storm signaling, among others, in the sequential future ACR group (Figure 1B). Together, these results suggest heightened epithelial and immune cell remodeling during ACR, accompanied by activation of cytokine-driven pathways that regulate epithelial barrier integrity and enhance expression of ciliary and cytoskeletal genes under inflammatory stress. Conclusions Transcriptomic profiling of lower airway samples revealed distinct epithelial and cytoskeletal gene signatures that may be associated with ACR. Enrichment of cilium- and microtubule-related pathways preceding rejection, and activation with RHO GTPase and cytokine signaling during ACR, suggest early and coordinated epithelial immune remodeling. This abstract is funded by: American Society of Transplantation (JGN)
Herrera et al. (Fri,) studied this question.