298 - Innate Lymphoid Cell Plasticity Drives Radiation-Induced Lung Injury

02:15pm - 02:25pm ET

Room 160

Presenter(s)

Jaeho Kim, MD, PhD - City of Hope, Duarte, CA

J. Kim¹, H. LI¹, R. Mannan², T. A. Doherty³, and H. M. McGee¹; ¹Department of Radiation Oncology, City of Hope National Medical Center, Duarte, CA, ²Department of Pathology, City of Hope National Medical Center, Duarte, CA, ³University of California, San Diego, San Diego, CA

Purpose/Objective(s):

Radiation-induced lung injury (RILI) is a major dose-limiting toxicity in lung cancer radiotherapy, yet the early immune mechanisms that initiate tissue remodeling after ablative irradiation (RT) remain incompletely defined. Innate lymphoid cells (ILCs) respond to tissue damage signals in their local microenvironment and do not express antigen receptors. We hypothesized that radiation-induced Type I IFN signaling promotes ILC2-to-ILC1 plasticity, exacerbating radiation-induced lung injury.

Materials/Methods:

A custom device was used to irradiate a 7mm diameter region in the left lung of mice to a dose of 60 Gy. Lungs were harvested at several time points after RT for flow cytometry of ILC subsets (CD45.2+Lineage-Thy1.2+), ELISA-based cytokine profiling, and histopathology to assess RILI. ILC2 were defined as ST2+ c-Kit-, ILC1 as ST2- c-Kit-, and ILC3 as ST2- c-Kit+. ILC-enriched lung immune cells underwent single cell RNA-seq with clustering and pseudotime analysis. RILI was quantified by a board-certified pathologist in C57Bl/6 WT, Rag2-/- and Rag2-/-Il2rg-/- (ILC-deficient) mice. ILC2s were sorted and adoptively transferred into Rag2-/-Il2rg-/- mice, and flow cytometry of ILC subsets and histopathologic analysis of RILI were performed. Sorted ST2+ ILC2s were stimulated in vitro with IFN-b, and ILC phenotype was characterized by flow cytometry. Anti-IFNAR1 antibody was injected to block type I IFN signaling in vivo after RT.

Results:

RT increased the weight of the left lung (mean 121mg vs. 82mg, p = 0.002) and induced marked inflammation at 2 weeks, similar in WT and Rag2-/- mice, but significantly reduced in Rag2-/- lL2rg-/- mice (inflammation score 3.6 vs. 4.3 vs. 1.7, p = 0.0011). ILCs accumulated in the lung post-RT and peaked at day 10. Total ILC numbers were relatively stable, yet ILC composition shifted after RT: ILC2s decreased while ILC1s increased (mean ILC1 percentage 66.2% vs. 20.0%, p < 0.0001; mean ILC2 percentage 16.8% vs. 73.5%, p < 0.0001), accompanied by a functional switch toward type 1 cytokines (increased IFN-? and TNF-a). Single cell RNA-seq identified an RT-expanded ILC1 cluster and a continuous pseudotime trajectory from ILC2 toward ILC1 after RT. Differentially expressed genes showed type I interferon-related response enrichment. ELISA revealed increased IFN-ß protein in lung tissue after RT. IFN-ß stimulation induced ILC2?ILC1-like conversion in vitro (characterized by ST2 loss, T-bet/IFN-? induction, and GATA3 suppression). Anti-IFNAR1 antibody injection post-RT significantly suppressed ILC1 expansion, partially restored ILC2s, and reduced RILI (mean histologic inflammation score 3.6 vs. 5.6, p = 0.0462).

Conclusion:

Type I IFN signaling promotes radiation-induced ILC plasticity and contributes to RILI. Targeting IFNAR1 may provide a novel immune-directed strategy to mitigate RT-induced tissue toxicity.