Immune checkpoint inhibitor-induced alterations in thymic gene expression and spatial distribution

Abstract

Immune checkpoint inhibitors (ICIs) represent a highly effective therapeutic approach in oncology, but their use is frequently associated with immune-related adverse events (irAEs). In our previous work, we showed that ICI treatment enhances the expression of inflammatory genes in the thymus. In parallel, clinical studies have reported severe cardiotoxicity in patients receiving ICI treatment for thymoma. Together, these observations point to a possible involvement of thymic function in the development of ICI-associated autoimmune toxicities, although the underlying mechanisms remain poorly understood.

The objective of the present study was to characterize thymic gene expression changes induced by ICI treatment using a preclinical mouse model.

Mice received a PD-1 inhibitor or vehicle control injection intraperitoneally, three times weekly for two weeks. Thymic gene expression was assessed using transcriptional profiling (RNA-seq) and further examined in a spatial context with RNAscope analysis.

In young male mice, differential gene expression was observed between ICI-treated and control groups. From the 212 differentially expressed genes we identified four targets (Cxcl9, Dock8, Ccl21a, Ets1), focusing on genes related to key leukocyte developmental processes, based on previous literature data. In ICI treated samples, Cxcl9 showed focal accumulation in the medulla. Dock8 was primarily detected in the cortex region, colocalizing with the T-cell marker Cd3e. Ccl21a, a marker of mTECI cells, was restricted to the medulla, while Ets1 was expressed in both regions. Notably, in the cortex, Ets1 was predominantly T-cell associated, whereas the ratio of Ets1-expressing T cells was much lower in the medulla.

Our findings show that ICI therapy induces significant transcriptional changes in the thymus, affecting key genes involved in leukocyte development and immune regulation. By mapping the expression of key target genes at the tissue level, we identified distinct spatial patterns that may provide explanation to ICI-related thymic immune regulation.