B28-25 Clonal Persistence of Cytotoxic T Cells Following Acute Lung Allograft Dysfunction in Lung Transplant Recipients

Abstract Rationale Expanded cytotoxic T cells mediate Acute Lung Allograft Dysfunction (ALAD), including Acute Cellular Rejection (ACR), increasing risk for later development of Chronic Lung Allograft Dysfunction (CLAD). Whether persistent T cell clones contribute to CLAD remains unclear. We hypothesized that clonally expanded T cells persist following ALAD/ACR, with gene expression programs driving tissue residency. Using single-cell RNA with concurrent T cell receptor sequencing, we investigated clonal persistence before and after ALAD/ACR episodes, characterized differentially expressed genes, and determined compositional shifts among expanded T cells. Methods Single-cell RNA and TCR sequencing was performed on 22 fresh transbronchial biopsies: 3 ACR (grade A1-A2), 2 ALAD (infection and aspiration), 1 acute lung injury (ALI), and 16 surveillance (No ACR/ALAD). Longitudinal sampling included patient 1 (grade A2 ACR with 4 follow-ups from 1-30 months post-ACR) and patient 2 (a surveillance 4 months prior to ALAD-infection, a follow-up surveillance 2 months post-ALAD, and grade A1 ACR occurring 5 months post-ALAD resolution). Data was integrated with Harmony, cell annotation via SingleR (historical data), clonal analysis with scRepertoire, and differential expression via DESeq2 (pseudobulk). Results Expanded cytotoxic T cell clonotypes (n 2) persisted across all biopsies in patient 1, spanning 1-30 months post-ACR, though their relative abundance declined over time (A). In patient 2, expanded T cell clonotypes identified during ALAD/ACR episodes showed substantial overlap with clonotypes present in pre-rejection surveillance biopsies (B). Expanded T cells exhibited upregulated tissue residency programs, including CXCR6, and increased HLA class II gene expression (HLA-DR, HLA-DP) compared to non-expanded cells. Cytotoxic (GZMB, NKG7) and activation (IFNγ) markers were also elevated in expanded cells (C). Expanded T cells were enriched for tissue-resident memory (Trm), effector (Teff), and exhausted (Texhaust) cell types compared to non-expanded (D). Conclusion Clonal persistence in two patients demonstrated that pathogenic cytotoxic T cell clonotypes exist prior to ALAD/ACR episodes, undergo expansion from pre-existing clonotypes rather than arising de novo during rejection, and may persist for years following ACR. These persistent expanded clonotypes represent a risk factor for CLAD development. The activation of tissue residency genes in expanded T cells suggests a mechanism for long-term allograft infiltration. Furthermore, upregulated HLA class II expression in expanded T cells indicates crosstalk with CD4+ T cells, potentially establishing a positive feedback loop that amplifies rejection. Together, these findings identify clonal persistence and tissue residency as therapeutic targets to prevent recurrent rejection and progression to CLAD in lung transplant recipients. This abstract is funded by: Heart Institute, Cincinnati Children’s Hospital Medical Center