Using fluorescence-activated cell sorting of PDGFRα+ fibroblasts from Lewis lung carcinoma (LLC GFP)–bearing lungs and single-cell RNA sequencing, the investigators identified seven transcriptionally distinct fibroblast clusters. One cluster was uniquely marked by high expression of cell adhesion molecule L1-like (Chl1) and was predominantly detected in tumor-associated lung tissue but largely absent from nonmalignant lung. Differential gene expression analysis showed this Chl1+ cluster expresses an immunomodulatory program that includes Timp1, Cxcl9, Cxcl10, H2-Ab1, Tgfb1 and Tnc. Reactome pathway analysis indicated enrichment for pathways facilitating interactions with cells of the adaptive immune system. The authors therefore termed this population imCAFs.
Immunofluorescence and flow cytometry confirmed CHL1 protein expression on this fibroblast subset and localized CHL1+ imCAFs primarily to the tumor border. Co-staining for canonical myofibroblast markers (such as tenascin-C and αSMA) showed minimal overlap with CHL1+ cells, supporting that imCAFs represent a distinct stromal population rather than a subset of canonical myCAFs. CHL1+ fibroblasts were also detected in genetically engineered Kras-driven lung adenocarcinoma models, indicating imCAFs are present across multiple murine LUAD models.
To assess spatial relationships within the tumor microenvironment, the authors combined single-cell and spatial transcriptomics and applied a machine learning mapping model. imCAFs localized to peritumoral regions where T cells are also enriched. Spatial cross-correlation analysis showed a high spatial correlation index between imCAFs and T cells, suggesting localized interactions.
Immunofluorescence microscopy with nearest-neighbor analysis revealed a particularly strong colocalization of CHL1+ imCAFs with FOXP3+ regulatory T (T reg) cells relative to conventional CD4+ (T conv) and CD8+ T cells. Deconvolution of spatial transcriptomic data with a reference-free modeling tool corroborated that T reg cells had the highest spatial correlation with imCAFs among T cell subsets.
Flow cytometry and transcriptional profiling indicated that T reg cells are enriched in tumor tissue compared with normal-adjacent lung or control lungs lacking tumors. Tumor-associated T reg cells exhibited elevated markers of proliferation and immunosuppressive function and were the most proliferative T cell subset within tumor-bearing lungs. These data suggest T reg cells in the tumor microenvironment are activated and primed to exert immunosuppressive effects.
Among the top differentially expressed genes in imCAFs was Cxcl9, a chemokine known to attract CXCR3-expressing T cells. In the tumor-bearing lung, imCAFs were the primary source of Cxcl9 relative to other cell types, including dendritic cells and macrophages. RT–qPCR confirmed elevated Cxcl9 expression in CHL1+ imCAFs compared with CHL1− CAFs.
Spatial and imaging analyses showed T reg cells preferentially localized to CXCL9-producing CHL1+ imCAFs. The number of CXCL9+ imCAFs correlated with local T reg cell abundance, whereas CXCL9 expressed by CHL1− cells did not correlate with T reg localization or numbers, indicating the imCAF context is critical for this recruitment.
Functional chemotaxis assays demonstrated that tumor-derived T reg cells migrate toward CXCL9 gradients to a significantly greater extent than tumor-derived conventional CD4+ or CD8+ T cells and greater than T cells from non-tumor tissues. Although Cxcl10 was also upregulated in imCAFs, CXCL10 gradients did not preferentially induce migration of tumor-infiltrating T reg cells in the assays reported.
The data indicate that T reg cells recruited to CXCL9-producing imCAFs express CXCR3 and comprise a hyper-suppressive subset. These CXCR3+ T reg cells accumulate at tumor borders, display enhanced proliferative and suppressive phenotypes compared with CXCR3− T reg cells, and are transcriptionally distinct from their CXCR3− counterparts. The CXCL9–CXCR3 signaling axis therefore appears to underpin selective recruitment and localization of this potent regulatory subset.
The source reports genetic loss-of-function experiments in which ablation of Cxcr3 in T reg cells or Cxcl9 in stromal cells reduced accumulation of T reg cells in tumors, enhanced activation of CD8+ T cells, and decreased tumor burden in mouse models. These perturbations link the imCAF-derived chemokine gradient to T reg recruitment and downstream effects on antitumor immunity and tumor growth.
Analysis of human non-small cell lung cancer samples identified CHL1+ fibroblasts with an imCAF-like transcriptional signature colocalizing with T reg cells. Elevated CHL1 expression in human tumors associated with reduced cytotoxic immune activity and decreased progression-free survival, supporting clinical relevance of the imCAF–CXCL9–CXCR3+ T reg axis reported in mouse models.
The study highlights a stromal mechanism whereby CHL1+ imCAFs shape an immunosuppressive niche by producing CXCL9 and recruiting hyper-suppressive CXCR3+ T reg cells to tumor borders. The reported genetic interventions that disrupt Cxcl9 or Cxcr3 reduced T reg accumulation, increased CD8+ T cell activation and lowered tumor burden, suggesting that targeting components of this axis may be a promising strategy to reprogram the tumor microenvironment and enhance antitumor immunity in lung adenocarcinoma.
Note: Specific experimental details, numerical values for some outcomes, and full methods are reported in the original source article.